Maintenance of genomic stability in cultured stem cells

ABSTRACT

The present application relates to methods and compositions for the generation of therapeutic cells having reduced incidence of karyotypic abnormalities. In several embodiments cardiac stem cells are cultured in an antioxidant-supplemented media that reduces levels of reactive oxygen species, but does not down regulate DNA repair mechanisms. In several embodiments, physiological oxygen concentrations are used during culture in order to increase the proliferation of stem cells, decrease the senescence of the cells, decrease genomic instability, and/or augment the functionality of such cells for cellular therapies.

RELATED CASES

This application is a continuation of co-pending U.S. patent applicationSer. No. 13/096,931, filed Apr. 28, 2011, which claims the benefit ofU.S. Provisional Application No. 61/330,251 filed on Apr. 30, 2010, theentire contents of each being expressly incorporated by referenceherein.

STATEMENT REGARDING GOVERNMENT SPONSORED GRANT

This invention was made with Government support under the ResearchProject Grant (R01HL083109) by the National Institutes of Health. TheUnited States Government has certain rights in this invention.

BACKGROUND

1. Field of the Invention

The present application relates generally to methods and compositionsfor generating genomically stable stem cells for the repair orregeneration of damaged cells or tissue. For example, in severalembodiments the methods and compositions disclosed herein may be usedfor the repair and/or regeneration of cardiac tissue. In particular,isolated cardiac cells are cultured in oxygen concentrations and/or inthe presence of antioxidant compositions that maintain an optimalbalance between reduced oxidative-stress induced DNA damage andfunctional DNA repair systems, thereby reducing genomic instability(e.g., DNA damage or karyotypic abnormalities) in the cultured cells.

2. Description of the Related Art

The scope of human disease that involves loss of or damage to cells isvast and includes, but is not limited to neurodegenerative disease,endocrine diseases, cancers, and cardiovascular disease. For example,coronary heart disease is presently the leading cause of death in theUnited States, taking more than 650,000 lives annually. According to theAmerican Heart Association, 1.2 million people suffer from a heartattack (or myocardial infarction, MI) every year in America. Of thosewho survive a first MI, many (25% of men and 38% of women survivors)will still die within one year of the MI. Currently, 16 millionAmericans are MI survivors or suffer from angina (chest pain due tocoronary heart disease). Coronary heart disease can deteriorate intoheart failure for many patients. 5 million Americans are currentlysuffering from heart failure, with 550,000 new diagnoses each year.Regardless of the etiology of their conditions, many of those sufferingfrom coronary heart disease or heart failure have suffered permanentheart tissue damage, which often leads to a reduced quality of life.

SUMMARY

Cell therapy, the introduction of new cells into a tissue in order totreat a disease, represents a possible method for repairing or replacingdiseased tissue with healthy tissue. However, cells generated for celltherapies have the potential to develop genomic abnormalities when beingprocessed for regenerative therapies. Such abnormalities could lead toreduced efficacy of the cell therapy or to neoplastic development at thetarget tissue. Accordingly, it is highly desirable to provide methodsand compositions for generating cells for cellular therapy that haveenhanced genomic stability.

In several embodiments, there is provided a method for reducing theincidence of karyotypic abnormalities in cardiac stem cells for use inthe repair or regeneration of cardiac tissue, comprising isolatingcardiac stem cells and culturing the isolated stem cells in a culturemedia supplemented with an antioxidant composition. In severalembodiments, the cells are isolated from healthy mammalian non-embryoniccardiac tissue, and then cultured.

In several embodiments there is provided a composition for reducing theincidence of karyotypic abnormalities in cultured cardiac stem cells,the composition comprising at least one peptide antioxidant, at leastone non-peptide antioxidant; and a culture media suitable for culturingcardiac stem cells, wherein the culture media is supplemented with theat least one peptide antioxidant and the at least one non-peptideantioxidant.

In several embodiments, there is provided a composition for reducing theincidence of karyotypic abnormalities in cultured cells, the compositioncomprising a culture media suitable for culturing cells, and at leastone antioxidant.

In several embodiments the at least one antioxidant is present in aconcentration ranging from about 0.1 to 200 μM, and functions to reducereactive oxygen species (ROS) to a level which decreasesoxidative-stress induced DNA damage in the cultured cells. In severalembodiments, the antioxidant composition comprises at least one peptideantioxidant and at least one non-peptide antioxidant. Although theconcentrations of the peptide antioxidant(s) and non-peptideantioxidant(s) may vary according to the embodiment (e.g., based on thecell type, the age of the source tissue, or other factors), in severalembodiments the at least one peptide antioxidant and the at least onenon-peptide antioxidant are present in an individual or combinedconcentration ranging from about 0.1 to 200 μM. In several embodiments,the antioxidant composition is suitable for reducing formation of ROSsuch as peroxides and free radicals, among others. In some embodiments,the reduced formation of ROS results in a level of ROS which decreasesoxidative-stress induced DNA damage, yet the resulting ROS levels arenot so low that markers of DNA repair mechanisms are significantlyreduced, and further the reduction does not significantly induce markersof DNA damage in the cardiac stem cells. Thus, in several embodiments,the balance of reduced ROS generation, non-reduced DNA repair mechanismsand non-induction of DNA damage reduces the overall incidence ofkaryotypic abnormalities in the cardiac stem cells. In severalembodiments, this is particularly advantageous, as karyotypicabnormalities reduce the percentage of usable cells in a pool of cellsto be used in cell therapy, but also present the risk of unwantedneoplastic growth (teratoma formation). Thus, in several embodiments themethods and compositions disclosed herein yield a greater number ofcells suitable for cell therapy (either in total number or based on apercent of useable cells) and yield cells that are safer for use in celltherapies. In several embodiments the markers of DNA repair mechanismscomprise one or more DNA repair enzymes selected from the groupconsisting of: ATM, ATR, Rad50, Rad51, Chk1, and Chk2 and in severalembodiments the markers of DNA damage comprise one or more of γ-H2AXfoci in cultured cells, γ-H2AX mRNA, or γ-H2AX protein. Other markers ofDNA repair or DNA damage are evaluated in other embodiments (e.g.,phosphorylation of H2AX, 7-hydro-8-oxo-2′-deoxyguanosineconcentrations). As a result of culturing cells in the cultureconditions and compositions provided in several embodiments, ROS levelsare reduced by at least 10%. In other embodiments, greater reductions inROS are achieved (e.g., at least 15%, at least 20%, or more). In stilladditional embodiments, a balance between reduced ROS generation,non-reduced DNA repair mechanisms and non-induction of DNA damaged isnot required to realize reductions in karyotypic abnormalities. Forexample, in some embodiments, alterations of one or more of theabove-referenced characteristics is sufficient to yield a reduction inkaryotypic abnormalities.

In several embodiments, the at least one peptide antioxidant is presentin a concentration ranging from about 0.1 to 200 μM. In severalembodiments, the at least one peptide antioxidant comprises glutathione.In some embodiments, glutathione can be supplemented by includingglutathione precursors in the culture media (for examplen-acetylcysteine, s-adenosylmethionine or whey protein). As such, thecultured cells not only have the glutathione in the media available, buthave precursor compounds present to increase the amount of glutathioneproduction in response to changing culture conditions.

Similarly, in several embodiments the at least one non-peptideantioxidant is present in a concentration ranging from about 0.1 to 200μM. In several embodiments the at least one non-peptide antioxidant isselected from the group consisting of thiols, vitamins and polyphenols.In some embodiments, these non-peptide antioxidants function toterminate chain oxidation reactions which are responsible, at least inpart, for generation of free radicals. In some embodiments, theseantioxidants “sacrifice” themselves by being oxidized, thereby sparingthe genetic material of the cell from damage. In several embodimentsvitamins are used as the non-peptide antioxidant, and may comprise oneor more vitamins selected from the group consisting of: vitamin A,vitamin E, and vitamin C. In several embodiments the various vitaminsare present in an individual or total concentration ranging from about 1to 150 μM. In still additional embodiments, other antioxidants may beused to supplement culture media. For example, enzymes such as catalase,superoxide dismutase and various peroxidases are used in someembodiments.

Other concentrations are used in some embodiments, depending on the celltype, the age of the cells, the time the cells have been in culture, thepassage number of the cell population. The various components of theantioxidant composition may, in some embodiments, be balanced toadvantageously tailor the composition to a particular set ofcharacteristics possessed by a particular cell population. For example,in some embodiments, an high passage cell population may benefit from agreater concentration of a peptide antioxidant as compared to anon-peptide antioxidant (or vice versa).

In several embodiments, cells are cultured with the antioxidantcomposition for about 24 hours. In some embodiments, shorter culturetimes are used (e.g., about 4-6 hours, about 5-10 hours, about 8-16hours, about 16 to 20 hours, and overlapping ranges thereof). In someembodiments, longer culture times are used (e.g., about 24-36 hours,about 36-48 hours, about 48-72 hours, or longer). In some embodiments,time can be varied depending on a variety of factors. For example, theage of the source tissue may be a factor in determining how long a cellneeds to be cultured for the antioxidant composition to be effective.The overall metabolic status of the cells may also be an importantvariable. Active cells may more effectively metabolize the antioxidantcompositions, thereby benefiting from the antioxidant effects describedherein. However, in some cases, too great a metabolic rate may overwhelmthe antioxidant composition, thereby requiring additional time inculture, increased concentrations of the compositions disclosed herein,or combinations of both.

In one embodiment, cardiac cells are cultured with an antioxidantcomposition comprising the peptide antioxidant glutathione in aconcentration ranging from about 0.1 to 20 μM, and a combination ofnon-peptide antioxidants comprising vitamin C and vitamin E, in aconcentration ranging from about 0.1 to 20 μM. In some embodiments,antioxidant compositions are used in conjunction with other methods toreduce the incidence of karyotypic abnormalities in cells. For example,in some embodiments, cells are cultured in an environment that moreclosely mimics the natural in vivo conditions for that cell (e.g.,physiologic oxygen concentrations), in conjunction with the use ofantioxidant compositions.

In several embodiments, there is provided a method of increasing theyield of stem cells in culture, comprising obtaining a population ofstem cells isolated from a source of tissue, restricting oxygenconcentrations in a culture environment to physiologic oxygenconcentrations, and culturing the stem cells in the restricted oxygenculture environment. In several embodiments, physiologic levels ofoxygen increase the rate at which the stem cells proliferate, therebyincreasing the yield of stem cells as compared to culture conditionsthat employ non-physiologic levels of oxygen. In several embodiments,the yield of stem cells is increased per unit weight of the sourcetissue as compared to the yield of stem cells cultured in conditionsthat employ non-physiologic concentrations of oxygen. In someembodiments, the per unit weight yield is increased by at least about 5%for a given time period of culturing. In some embodiments, the per unitweight yield is increased by at least about 20% for a given time periodof culturing. In several embodiments, greater increases in per unitweight yield are achieved (e.g., at least 25%, 30%, 35%, 40%, 50%, orgreater). In several embodiments the source tissue is cardiac tissue andthe stem cells are cardiac stem cells.

Advantageously, in several embodiments, the increased yield reduces theamount the amount of time that the stem cells are cultured in order toreach a certain population as compared to the amount of time stem cellsare cultured in non-physiologic concentrations of oxygen in order toreach the certain population. For example, in some embodiments, theincreased yield reduces the amount the amount of time required forculturing by 20%. In additional embodiments, the increased yield reducesthe amount the amount of time required for culturing by 50%. Suchincreased yield (and reduced culture time) are particularly advantageousin some embodiments, wherein the cells are to be used for therapy(reduced time from collection to therapy), or in generating a cell bank(reduced time to generate a sizeable bank for future therapy). Moreover,in either in autologous or allogeneic transplant scenarios, the reducedtime to generate a given population size reduces the time between tissuecollection and subsequent therapy. In some allogeneic contexts, thistime is negligible, because a cell bank can be generated prior to theneed for any other subject to receive therapy.

In several embodiments, there is provided a method for increasing thefunction of the cardiac tissue of a subject having damaged or diseasedcardiac tissue, comprising obtaining a population of cardiac stem cellsfor administration to the subject, wherein the cardiac stem cells areharvested from donor cardiac tissue and expanded in a cultureenvironment comprising oxygen concentrations restricted to physiologicoxygen concentrations (to generate an expanded population of cardiacstem cells), and administering at least a portion of the expandedpopulation of cardiac stem cells to the subject. In several embodimentsthe administered cardiac stem cells engraft into the cardiac tissue ofthe subject to a greater degree than cardiac stem cells expanded innon-physiologic concentrations of oxygen. In several embodiments theadministered cardiac stem cells survive in the cardiac tissue of thesubject to a greater degree than cardiac stem cells expanded innon-physiologic concentrations of oxygen. In still additionalembodiments, the greater degree of engraftment and/or survival lead toincreased cardiac function in the subject.

In several embodiments, there is provided a method for enhancing theefficacy of cardiac stem cell therapy, comprising obtaining a populationof cardiac stem cells for administration to a subject in need of cardiacstem cell therapy due to damaged or diseased cardiac tissue wherein thecardiac stem cells are harvested from donor cardiac tissue and expandedin a culture environment comprising oxygen concentrations restricted tophysiological oxygen concentrations to generate an expanded populationof cardiac stem cells, and administering at least a portion of theexpanded population of cardiac stem cells to the subject.

In several embodiments the physiologic oxygen concentrations are betweenabout 1% to about 8%, about 2% to about 7% about 3% to about 6%, about4% to about 5%, and overlapping ranges thereof. In one embodiment, theoxygen concentration ranges from about 4% to about 7%. In severalembodiments, the oxygen concentrations are tailored to a particular celltype. For example, depending on the region of tissue from which apopulation of cells originated (e.g., a tissue having low oxygenconcentrations in vivo versus a tissue having high oxygen concentrationsin vivo) oxygen concentrations can be adjusted to be appropriatelyphysiologic for that cell type. Even within a particular organ, thedegree of oxygenation may vary. Tissue oxygen concentrations are readilydiscerned by one of ordinary skill in the art and can thus be used totailor the methods disclosed herein to generate greater numbers ofcells, more genetically stable cells, and/or cells with increasedfunctionality.

In several embodiments, the methods provided comprise culturing cells inphysiologic oxygen concentrations to reduce the incidence of karyotypicabnormalities in the cultured as compared to cells cultured innon-physiologic concentrations of oxygen. In some such embodiments,culturing in physiologic oxygen concentrations reduces the incidence ofaneuploidy in the cultured cells as compared to cells cultured innon-physiologic concentrations of oxygen. In some embodiments, DNAstrand breaks are reduced. In some embodiments, combinations ofreduction in karyotypic abnormalities, aneuploidy, and DNA strand breaksare reduced. In several embodiments the cultured cells are cardiac stemcells. In some embodiments, the cells are for use in allogeneic cardiaccell therapy. In other embodiments, the cells are for use in autologoustherapy. In several embodiments, the cardiac stem cells comprisecardiospheres, cardiosphere derived cells, or a subsequent generation ofcardiospheres. In some embodiments, the cardiac stem cells are suitablefor administration of a subject having damaged or diseased cardiactissue.

In several embodiments, the administration of the cardiac stem cellscultured in physiologic oxygen to a subject results in increasedengraftment into the cardiac tissue of the subject as compared toengraftment of cardiac stem cells cultured in non-physiologicconcentrations of oxygen. In several embodiments, the administration ofthe cardiac stem cells cultured in physiologic oxygen to a subjectresults in one or more of increased myocardial viability, increased wallthickness, and lower left ventricular volume in the cardiac tissue ofthe subject as compared to that resulting from administration of cardiacstem cells cultured in non-physiologic concentrations of oxygen. Inseveral embodiments, increased function due to administration of cellscultured in physiologic oxygen concentrations is realized as an improvedleft ventricular ejection fraction (by at least 5% as compared toincreased function due to administration of cardiac stem cells expandedin non-physiologic concentrations of oxygen). In several embodiments,the administration of the cardiac stem cells cultured in physiologicoxygen to a subject results in greater survival of the cells in thecardiac tissue of the subject to a greater degree than cardiac stemcells expanded in non-physiologic concentrations of oxygen

In several embodiments, a method for reducing the incidence ofkaryotypic abnormalities in cardiac stem cells for use in the repair orregeneration of cardiac tissue is provided. In one embodiment, themethod comprises isolating cardiac stem cells and culturing the isolatedstem cells in a culture media supplemented with an antioxidantcomposition. In one embodiment, the method comprises combining cellssusceptible to chromosomal damage with an antioxidant composition at aconcentration suitable to reduce the incidence of chromosomal damage inthe cells, when, for example, the cells are administered to a mammal. Inone embodiment, the antioxidant composition is provided at aconcentration that reduces free radical damage while still permitting(or without substantially impairing) the function of one or more of thecell's endogenous repair mechanisms.

In several embodiments, a composition for reducing the incidence ofkaryotypic abnormalities in cultured cardiac stem cells is provided. Inone embodiment, the composition comprises at least one peptideantioxidant, at least one non-peptide antioxidant, and a culture mediasuitable for culturing cardiac stem cells that is supplemented (orsuitable to supplementation) with the at least one peptide antioxidantand the at least one non-peptide antioxidant. In one embodiment, theantioxidant composition comprises, consists or consists essentially ofone or more non-peptide antioxidants. In another embodiment, theantioxidant composition comprises, consists or consists essentially ofone or more peptide antioxidants.

In several embodiments, a composition for reducing the incidence ofkaryotypic abnormalities in cultured cells is provided. In oneembodiment, the composition comprises a culture media suitable forculturing cells and at least one antioxidant present in a concentrationranging from about 0.1 to 200 μM. In some embodiments, the cells arestem cells. In some embodiments, the cells are cardiac stem cells.

In several embodiments, a method for reducing cellular and/or genetic(e.g., karyotypic) abnormalities in cells cultured in a medium isprovided. In one embodiment, the method comprises contacting the cellswith one or more of the compositions disclosed herein. In anotherembodiment, the method comprises culturing the cells in a hypoxicenvironment and, optionally, with an antioxidant composition accordingto several embodiments disclosed herein.

In several embodiments, the cells are isolated from healthy mammaliannon-embryonic cardiac tissue. In some embodiments, the cells areisolated from healthy mammalian non-embryonic non-cardiac tissue. Insome embodiments, the cells are isolated from embryonic tissue.

In several embodiments, the antioxidant composition reduces reactiveoxygen species to a level which decreases oxidative-stress induced DNAdamage, but does not significantly impair DNA (and/or other cellular)repair mechanisms. In one embodiment, antioxidant compositions accordingto several embodiments disclosed herein reduce oxidative-stress induceddamage without adversely affecting the cell's own repair mechanisms bymore than 1%, 5%, 10%, 25%, or 50%.

In several embodiments, the reduction in reactive oxygen species doesnot significantly induce DNA damage (as evidenced by markers of DNAdamage) in stem cells, such as cardiac stem cells. In severalembodiments, the balance of reduced reactive oxygen species, non-reducedDNA repair mechanisms and non-induction of DNA damage reduces theincidence of karyotypic abnormalities in cells, including stem cells(such as cardiac stem cells).

In some embodiments, at least one peptide antioxidant is selected fromthe group consisting of enzymes, proteins, peptides. In someembodiments, at least one peptide antioxidant comprises glutathione. Insome embodiments, glutathione is present in a concentration ranging fromabout 1 to 150 μM. In some embodiments, glutathione is present in aconcentration ranging from about 1 to 50 μM. In some embodiments,glutathione is present in a concentration ranging from about 0.1 to 20μM.

In some embodiments, at least one non-peptide antioxidant is selectedfrom the group consisting of thiols, vitamins and polyphenols. In someembodiments, at least one non-peptide antioxidant comprises one or morevitamins. In some embodiments, the vitamins comprise one or more ofvitamin A, vitamin E, and vitamin C. In some embodiments, the vitaminsare present in an individual or total concentration ranging from about 1to 150 μM. In some embodiments, the vitamins are present in anindividual or total concentration ranging from about 1 to 50 μM. In someembodiments, the vitamins comprise vitamin C and vitamin E. In someembodiments, vitamin C and vitamin E are present in an individual ortotal concentration ranging from about 1 to 50 μM. In some embodiments,vitamin C and vitamin E are present in a concentration ranging fromabout 0.1 to 20 μM.

In some embodiments, the antioxidant composition comprises, consists of,or consists essentially of glutathione at a concentration ranging fromabout 0.1 to 20 μM and vitamin C and vitamin E, which are each presentin a concentration ranging from about 0.1 to 20 μM.

In several embodiments, cells are exposed to antioxidant compositionsand/or hypoxic conditions to cause a reduction in reactive oxygenspecies of at least 10% as compared to cells cultured in 20% oxygen. Insome embodiments, the reactive oxygen species are reduced by at least50% as compared to cells cultured in 20% oxygen. In some embodiments,the reactive oxygen species are reduced by about 60%-70% (e.g., 65%) ascompared to cells cultured in 20% oxygen. In some embodiments, areduction in reactive oxygen species of at least 10%, 25%, 50% or 75% isachieved as compared to cells cultured in hyperoxic conditions. In oneembodiment, the use of antioxidant compositions disclosed herein reduceskaryotypic damage when cells are cultured in about 20% oxygen, orhigher. In one embodiment, the combined use of antioxidant compositionsand hypoxic conditions result in a synergistic effect (e.g., enhancedreduction of cellular/DNA damage).

In several embodiments, the markers of DNA repair mechanisms compriseone or more DNA repair enzymes. In some embodiments, DNA repair enzymesare selected from the group consisting of ATM, ATR, Rad50, Rad51, Chk1,Chk2, or combinations thereof. In several embodiments of the invention,compositions or hypoxic conditions disclosed herein, do not impair DNArepair mechanism at by more than 0% (e.g., no effect), 2%, 5%, 10%, 20%,30%, and 50%. DNA repair mechanisms include, but are not limited to,base excision, nucleotide excision and mismatch repair. In oneembodiment, compositions or hypoxic conditions disclosed herein maintainor up-regulate at least one repair mechanism while down-regulatinganother repair mechanism, wherein the overall impairment of DNA repairis no more than 0%, 2%, 5%, 10%, 20%, 30%, or 50%. In one embodiment,compositions or hypoxic conditions disclosed herein do not impair baseexcision, nucleotide excision or mismatch repair. In another embodiment,compositions or hypoxic conditions disclosed herein do not impair atleast two of base excision, nucleotide excision or mismatch repair. Inone embodiment, compositions or hypoxic conditions disclosed hereinreduce the incidence of karyotypic abnormalities in cells by more than10%, 25%, 50%, 75%, or 95% as compared to control cells.

In several embodiments, the markers of DNA damage comprise one or moremarkers of DNA double-strand breaks. In some embodiments, the markers ofDNA double-strand breaks comprise γ-H₂AX foci in cultured cells, γ-H₂AXmRNA, or γ-H₂AX protein, or combinations thereof.

In several embodiments provided herein, the isolated cells (e.g., stemcells) are cultured for at least about 24 hours, 3 days or 7 days. Insome embodiments, the isolated cells (e.g., stem cells) are cultured fora period of one to 6 months (e.g., 1, 2, 3, 4, 5, or 6 months).

In several embodiments, a method for assessing the risk of neoplasticdisease in a subject is provided. In one embodiment, the methodcomprises obtaining a blood or tissue sample from the subject andmeasuring the concentration of one or more reactive oxygen species inthe sample. In some embodiments, the risk for neoplastic disease isgreater if the level of reactive oxygen species is sufficiently high toinduce oxidative DNA damage in the cells of the subject and/or if thelevel of reactive oxygen species is sufficiently low to promotedown-regulation of DNA repair mechanisms in the cells of the subject.Reduction in the risk of neoplastic disease is achieved in severalembodiments of the invention by, for example, treating subjects withcells cultured in compositions or conditions disclosed herein.

In several embodiments, a method for assessing the risk of neoplasticdisease in a subject is provided. In one embodiment, the methodcomprises analyzing the subject's tissue for evidence of karyotypicabnormalities (e.g., DNA damage) and correlating the damage with thesubject's antioxidant levels. Optionally, the subject is then treated toreduce or increase antioxidant levels as needed. In one embodiment,evidence of damage is determined by analyzing markers of DNA damage, DNAdamage-associated mRNA expression levels, and/or DNA damage-associatedprotein expression levels. In one embodiment, the risk for neoplasticdisease increases as the incidence of DNA damage increases, as theexpression of DNA damage-associated mRNA increases, and/or as theexpression of DNA damage-associated protein increases. In someembodiments, DNA damage is measured by identifying γ-H₂AX foci, bymeasuring γ-H₂AX mRNA expression levels, and/or by measuring γ-H₂AXprotein expression levels.

In several embodiments, a method for optimizing the amount of anantioxidant-containing supplement administered to a subject is provided.In one embodiment, the method comprises measuring the concentration ofone or more reactive oxygen species in the subject's sample (e.g.,tissue samples, skin scrapings, blood, isolated cells, saliva, urine,other bodily fluids, etc.) and the incidence of DNA damage in cellsisolated from the sample. In some embodiments, in addition to, orinstead of, ascertaining DNA damage, the expression of DNA repairenzymes in cells isolated from the sample is measured. In oneembodiment, the method further comprises treating the patient with anantioxidant composition, and optionally adjusting the composition tobalance the concentration of reactive oxygen species in the subject withthe subject's endogenous DNA repair mechanism(s), as determined by, forexample, expression of DNA repair enzymes or other markers of DNArepair. In several embodiments, compositions (e.g., supplements)comprising an optimal amount of antioxidant(s) are provided that areformulated to decrease oxidative-stress induced DNA damage withoutnegatively impacting (wholly or partially) DNA repair mechanisms in thesubject. In one embodiment, therapeutic antioxidant compositions thatbalance oxidative damage with endogenous DNA repair mechanisms areprovided based on a subject's individual antioxidant and/or karyotypicprofile. In some embodiments, the invention comprises determining γ-H₂AXfoci to gauge risk of chromosomal damage (and neoplasm) and/or tooptimize antioxidant supplements.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts histograms summarizing karyotyping data for the variousculture conditions utilized in the Examples.

FIGS. 2A-2C depict representative karyotypes made from CDCs cultured invarious conditions.

FIGS. 3A-3D depict CDC proliferation data. FIGS. 3A, 3B, and 3C depictrepresentative microscopic images of CDCs cultured for 36 days invarious conditions (A=20% O₂; B=20% O₂ plus antioxidant supplement at1:1000 dilution; C=20% O₂ plus custom antioxidant cocktail at 100 μM).FIG. 3D depicts that no significant differences in CDC proliferationwere detected among the various culture conditions.

FIGS. 4A and 4B depict ATP and mitochondrial membrane potential data.

FIG. 4A depicts intracellular ATP concentrations and FIG. 4B depictsmitochondrial membrane potential of CDCs cultured in various conditions.

FIGS. 5A-5D depict intracellular ROS concentrations in humancardiosphere-derived cells after 24 hours culture under 20% O₂ withdifferent concentrations of antioxidants (5A and 5B), catalase (5C), andhydrogen peroxide (H₂O₂; 5D).

FIGS. 6A-6D depict flow cytometry assessments of intracellular ROSlevels in human CDCs cultured for 24 hours with different concentrationsof antioxidants (6A and 6B), catalase (6C), or hydrogen peroxide (H₂O₂;6D).

FIGS. 7A-7D depict ROS and DNA damage data. FIG. 7A depictsintracellular ROS concentration in human cardiosphere-derived cellsafter long-term culture under 20% O₂, 20% O₂, or under 20% O₂ with addedantioxidants. FIG. 7B depicts intracellular ROS concentrations afterculturing in hypoxic conditions or with the addition of antioxidants.FIG. 7C depicts γ-H₂AX foci in CDCs cultured in the indicatedconditions. FIG. 7D depicts quantitation of γ-H₂AX foci.

FIGS. 8A-8D depict representative microscopic images demonstratingγ-H₂AX foci in CDCs cultured with antioxidants (8A and 8B); catalase(8C) and hydrogen peroxide (H₂O₂; 8D).

FIGS. 9A-9D depict the bi-phasic DNA damage dose-response in human CDCsand embryonic stem (ES) cells cultured with varying amounts ofanti-oxidants (9A and 9B), catalase (9C) and hydrogen peroxide (H₂O₂;9D).

FIGS. 10A-10D depict ATM protein levels in human cardiosphere derivedcells after 24 hours of culture under 20% O₂ with antioxidants (10A and10B), catalase (10C), and hydrogen peroxide (H₂O₂; 10D).

FIGS. 11A-11D depict representative western blot analysis of theexpression of various DNA repair-related factors after culturing under20% O₂ with antioxidants (11A and 11B), catalase (11C), and hydrogenperoxide (H₂O₂; 11D).

FIGS. 12A-12E depict semi-quantitative histograms of western blot datafor the expression of ATR (FIG. 12A), Rad50 (FIG. 12B), Rad51 (FIG. 12C)Chk1 (FIG. 12D), and Chk2 (FIG. 12E).

FIGS. 13A and 13B depict DNA repair data. FIG. 13A depicts proteinexpression and quantitation of ATM from human cardiosphere-derived cellsafter 1-2 months long-term culture under the indicated conditions. FIG.13B depicts protein expression of various DNA repair-related factors inhuman cardiosphere-derived cells after 1-2 months long-term cultureunder different conditions.

FIG. 14 depicts a schematic of the possible interactions betweenintracellular ROS levels and genomic stability.

FIG. 15 depicts the difference in growth between culturing CDCs in 20%versus 5% O₂.

FIG. 16 depicts the number of euploid cells, aneuploid cells, and cellswith DNA breaks or translocations for 13 CDC lines divided and culturedin 20% or 5% oxygen.

FIG. 17 depicts a significant improvement in the efficacy of CDCscultured in physiologic oxygen concentrations as compared to thosecultured in room oxygen.

FIGS. 18A-18D depict the growth and proliferation of cardiac stem cellsin 5% O₂ and 20% O₂. Panel A shows representative images showing thatcell outgrowth from ‘explants’ (red arrow) was much faster in 5% O₂(right) than in 20% O₂ (left). The number of cells harvested (Panel C)was more than two-fold higher in 5% O₂ than in 20% O₂, although theamount of starting material was equivalent. Panel B depicts CDCs atearlier passages (shown in passage #2) showing no differences in eithermorphology or proliferative activity (Panel D) under 5% O₂ and 20% O₂,although greater proliferation was observed in cells expanded under 5%O₂ at later passages.

FIGS. 19A-19B depicts analysis of chromosomal abnormalities. Panel Aindicates fewer aneuploid cells in CDCs cultures expanded in 5% O₂ ascompared to 20% O₂. Panel B shows that the percentages of aneuploidcells were also decreased in 5% O₂ culture.

FIGS. 20A-20F depict analysis of cell senescence. Compared with 20% 02culture, cell senescence of CDCs was improved under 5% O₂ culture, byflow cytometry for p16^(INK4A) (panels A and D), immunostaining fortelomerase activity (panels B and E), and senescence-associatedb-galactosidase staining (panels C and F).

FIGS. 21A-21F depict analysis of intracellular ROS, DNA damage, andresistance to oxidative stress. Panel A shows that the levels ofintracellular ROS are lower in CDCs expanded in 5% O₂ when compared withthose in 20% O₂. Panel C depicts DNA damage as evidenced by theformation of γ-H₂AX foci. DNA damage was lower in CDCs expanded in 5% O₂than in 20% O₂ (Panel D). Panel E shows representative images ofTUNEL-positive (red) CDCs after 24 h exposure to 100 mM H₂O₂. The numberof apoptotic cells (panel F) was lower in CDCs expanded in 5% O₂ than20% O₂.

FIGS. 22A-22E depicts analysis of cell engraftment, cardiac functionalrecovery, and their relationships. Panel A depicts quantitative data onthe survival rate of human CDCs 24 h and 7 days after implantation intomice-infarcted heart. Panel B shows cells positively stained by HNA aremore frequently observed in mice 3 weeks after implantation with CDCsexpanded in 5% O₂ than in 20% O₂. Panel C shows quantitative data forcell engraftment (% nuclei) in the infarcted heart. Panel D shows thatleft ventricular ejection fraction (LVEF) at baseline does not differamong groups, indicating a similar infarct size to begin with. After 3weeks, the LVEF was higher in mice implanted with CDCs expanded in 5% O₂than in 20% O₂, although the LVEF was also higher in mice implanted withCDCs expanded in 20% O₂ than in controls with PBS injection only. PanelD indicates that the engraftment of human CDCs (% nuclei) within theinfarcted hearts of mice is strongly correlated with the absolute valuesof LVEF at 3 weeks.

FIGS. 23A-23G depict histological assessments of infarct size andventricular morphology. Representative images of Masson's staining showthat, compared with an infarcted heart receiving PBS injection only(panel A), the infarct size was much smaller in a heart that hadreceived CDCs expanded in both 5% O₂ (panel C) and 20% O₂ (panel B).Quantitative analyses of viable myocardium (panel D), LV wall thickness(panel E), LV chamber area (panel F), and LV total area (panel G) showthat better therapeutic efficiency was achieved by the implantation ofCDCs expanded in 5% O₂ than in 20% O₂, although a significantimprovement was also observed by the implantation of CDCs expanded under20% O₂ when compared with the control treatment with PBS injection only.

FIG. 24 depicts a representative karyotype showing aneuploidy (in thiscase, trisomy 8) in twice-passaged cardiac-derived cells expanded under20% O₂.

FIGS. 25A-25D depict cell phenotype and in vitro myogenicdifferentiation. Panel A shows flow cytometry data shows no significantdifference in the proportion of c-kit⁺ stem cells in cardiac-derivedcells expanded in 5% O₂ versus 20% O₂ (quantification in panel C). PanelB shows similar expression of troponin T was also observed in the twogroups (quantification in panel D).

FIGS. 26A-26D depict analysis of the expression of adhesion moleculesand c-Myc (panel A shows Western blot protein expression data). There isno significant difference in the expression levels of integrin-α₂ (panelB), laminin-β₁ (panel C), or c-Myc (panel D) in cardiac-derived cellsexpanded in 5% O2 versus 20% O₂.

FIGS. 27A-27F depict a comparison of in vitro production of growthfactors between cardiac-derived cells expanded in 5% O₂ and 20% O₂. Theconcentrations of angiopoietin-2 (panel A), bFGF (panel B), HGF (panelC), IGF-1 (panel D), SDF-1 (panel E), and VEGF (panel F) measured byELISA showed no significant difference in conditioned medium culturewith hypoxic stimulation.

FIGS. 28A-28B depict an in vitro angiogenesis assay. Panel A shows theformation of capillary-like tubes in extracellular matrix fromcardiac-derived cells expanded in 5% O₂ and 20% O₂ did not differ due tohypoxic stimulation. Panel B shows the quantitative summary data fortube length.

FIGS. 29A-29D depict immunostaining analysis showing that some humanCDCs (identified by expression of human nuclear antigen, HNA) expandedin 5% O₂ (panel A) and 20% O₂ (panel B) expressed α-sarcomeric actin(α-SA), which is indicative of myogenic differentiation 3 weeks afterimplantation into infarcted hearts of mice. Image insets (panels C andD) show that the human nuclei are actually within cardiomyocytes.

FIGS. 30A-30B depict immunostaining analysis shows some humancardiac-derived cells (CDCs, identified by human nuclear antigen, HNA)expanded in 5% O₂ (panel A) and 20% O₂ (panel B) expressed smooth muscleactin (SMA) 3 weeks after implantation into infarcted hearts of mice.

FIGS. 31A-31B depict immunostaining analysis shows some humancardiac-derived cells (CDCs, identified by human nuclear antigen, HNA)expanded in 5% O₂ (panel A) and 20% O₂ (panel B) expressed theendothelial marker von Willebrand factor (vWF) 3 weeks afterimplantation into infarcted hearts of mice.

DETAILED DESCRIPTION

In several embodiments described herein, methods of reducing theincidence of karyotypic abnormalities in cultured stem cells for use inthe repair or regeneration of cardiac tissue are provided. In severalembodiments, compositions that reduce the incidence of karyotypicabnormalities in the culturing of stem cells are provided. Suchembodiments are advantageous because chromosomal abnormalities have beenfound in an unexpectedly large percentage of stem cells isolated,cultured, and expanded for use in cellular therapies. For example,chromosomal abnormalities have been found in up to 50% of long-termcultured human embryonic stem (ES) cells. Normal embryonic stem cells,which are typically derived from an early stage embryo, have thepotential to develop into any type of cell in the body. In someinstances, unplanned growth of one cell type in a distinct type oftissue may result in the formation of teratomas. Undesirable neoplasticgrowth may be potentiated if ES cells with chromosomal abnormalities areused in therapy.

In contrast to ES cells, adult stem cells generally develop into celltypes related to the tissue from which the stem cells were isolated.However, the potential for undesirable neoplastic growth still exists,particularly if adult stem cells with chromosomal abnormalities are usedin therapy. In fact, G-banding karyotype analysis of primarycardiosphere-derived cells (CDCs, a heart-derived mixed-cell populationrich in cardiac stem cells) revealed that ˜30% of preliminary CDCproduction runs resulted in cells with chromosomal abnormalities (SeeTable 1). In addition to potential neoplastic growth, genomicalterations of stem cells may impair therapeutic potency of administeredcells. Thus, several embodiments of the present invention areparticularly beneficial because they enhance the therapeutic potency(e.g., efficacy and/or viability, etc.) of the administered cells.

TABLE 1 Summary of Chromosomal Abnormalities Samples Karyotype 2424/p346, XX[20] 2377/p3 46, XX[20] 2404/p3 46, XY[20] 2482/p4 46, XY[20]R071215/p3 47, XY + 8[3]/47, XY + 18[3]/46, XY[44] R071215/p6 47, XY +8[2]/46, XY[48] R071212/p6 46, XY[50] R071214/p4 47, XY + 2[4]/46,XY[46] R071214/p5 47, XY + 2[6]/46, XY[44] R071214/p6 47, XY + 2[13]/46,XY[7] 6-1-1/p5 46, XY[20] 6-1-3/p5 46, XY[20] 10-1/p2 46, XX[20] CSB9/p446, XY, inv(9)(p11; q13) [20] CSB11/p3 46, XY[20] CSB3/p3 45, X −Y[6]/46, XY[14] BX13/p4 46, XY[20] BX14/p3 46, XX[20] BX15/p3 47, XY +8[20] BX16/p4 45, X − Y[7]/47, XY + i(8)(q10)[6]/47, XY + 8[1]/46, XY[6]BX17/p2 45, X − Y[3]/46, XY[17] BX18/p3 46, XY[20] BX19/p3 46, XY[20]BX20/p2 46, XY[20] BX21/p2 46, XY[20] BX22/p1 46, XY[20] BX23/p1 46,XY[20] BX23/p2 46, XY[20] BX24/p1 45, X − Y[10]/47, XY + 18[2]/46, XY[8]BX27/p1 47, XY + 8[6]/45, X − Y[3]/47, XY + Y[2]/46, XY[9] BX27/p2 47,XY + 8[14]/46, XY[6] BX43/p0 46, XY[20] BX45/p0 46, XY[20] BX46/p0 46,XY[20] BXJ4/p0 45, −Y, t(X; 11)(p10; p10)[11]/45, X, −Y[3]/46, XY[6]BX45/p1 46, XY[20] BX46/p1 47, XY + 8[4]/46, XY[16] BXJ4/p1 45, −Y, t(X;11)(p10; p10)[19]/46, XY[1] BX34/p1 46, XY[20] BX40/p2 46, XY[20]BXJ2/p1 46, XX[20]

Reactive oxygen species (ROS) are reactive molecules that contain theoxygen atom. The superoxide ion (O₂—) and peroxides (e.g., hydrogenperoxide, H₂O₂) are well known ROS. The superoxide ion leaks from activemitochrondria and is converted to H₂O₂. Cellular enzymes such ascatalase and superoxide dismutase act on H₂O₂ to form hydrogen andwater; however these reactions are not efficient enough to remove allthe H₂O₂. ROS molecules are typically highly reactive due to thepresence of unpaired valence shell electrons. While ROS are formed undernormal circumstances where oxygen is metabolized, and may play importantroles in cell signaling, during times of environmental or metabolicstress (e.g., UV or heat exposure, ionizing radiation, hypoxia,ischemia, etc.) ROS levels may increase, possibly resulting in damage tointracellular structures or induction of programmed cell deathmechanisms. The various pathways that generate ROS are known asoxidative stress. Increases in oxidative stress have the potential todamage RNA, DNA, or protein that, if uncorrected, may lead tochromosomal abnormalities. Chromosomal abnormalities, in turn, can leadto poorly functioning or malfunctioning cells, uncontrolledproliferation of cells, or apoptosis, among other outcomes.

Because cells will fail to function or function improperly if oxidativestress corrupts the integrity or accessibility of a cells genome,mammalian cells have developed a variety of innate DNA repairmechanisms. In some instances, cells will use the unmodifiedcomplementary DNA strand to recover any genetic information that is lostdue to oxidative stress. If both strands of DNA are damaged, the cellmay allow polymerases to replicate DNA through the site of the lesion inorder to replicate essential DNA sequences. However, none of theserepair mechanisms is completely free of errors. Thus, the properfunction and longevity of a cell is a balance of the oxidative stress acell experiences and the ability of one or more DNA repair mechanism tomaintain the cell's genome in normal working order.

Often, cells to be used in research or clinical applications arecultured in media equilibrated with 95% air and 5% CO₂ (˜20% O₂).Certain cells thrive in such an environment, as the oxygen concentrationmimics what those cells would be exposed to in vivo. However, in thecase of many stem cell varieties, depending on the tissue, cellularoxygen concentrations may be as low as ˜1-5% in the in vivophysiological microenvironment. Exposure of stem cells to anon-physiological hyperoxic state in culture may lead to oxidativestress, which, as discussed above, may induce ROS formation, DNA damage,and/or genomic instability. Genomic instability may be manifest inseveral ways, including karyotypic abnormalities, low viability cells,cells prone to neoplastic formation and the like. As used herein, theterms “physiologic oxygen concentrations” and “physiological oxygenconcentrations” shall be given its ordinary meaning and shall also referto oxygen concentrations ranging from about 1% to about 8% oxygen. Insome embodiments oxygen concentrations in which the CDCs (or othercardiac stem cells) are cultured range from about 1% to about 2%, about2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% toabout 6%, about 6% to about 7%, and overlapping ranges thereof. Inseveral embodiments physiologic oxygen conditions are maintainedthroughout the entire culture process, while in other embodiments,physiologic oxygen conditions are used in only a portion of the cultureprocess.

Therefore, in several embodiments, methods and compositions for reducingthe incidence of karyotypic abnormalities in cultured stem cells for usein the repair or regeneration of tissue are provided. In severalembodiments, use of the compositions and methods disclosed herein permitthe use of hyperoxic cell culture conditions. In several embodiments,cardiac tissue is repaired or regenerated. Other types of tissue (e.g.,kidney, lung, liver pancreas, spleen, bone, bone marrow, muscle tissue,vascular tissue, nervous tissue, skin, etc.) are repaired or regeneratedin other embodiments. In several embodiments the method comprisesproviding a culture media supplemented with an antioxidant cocktail(discussed in more detail below) that is capable of inducing a balancebetween the formation of ROS and the ensuing DNA damage and the innateDNA repair mechanisms of the cultured cells, thereby reducing in theincidence of karyotypic abnormalities. In some embodiments, the methodoptionally includes culturing the cells in a “hypoxic” environmentrelative to standard 20% O₂ cell culture conditions (e.g., oxygenconcentrations ranging from about 1% to about 8%). In severalembodiments, such culture conditions improve the viability of thecultured stem cells. In some embodiments, short term viability isimproved, while in some embodiments, long-term viability is improved. Insome embodiments, both short and long-term viability is improved. Assuch, in several embodiments, the proliferation rate of the cells inculture is increased. However, due to the normoxic (vis-à-vis the normalcellular environment) conditions, limited genetic alterations occur. Insome embodiments, this is particularly advantageous because the rate ofcell expansions reduces the amount of time needed to reach a certainpopulation of cells. In some embodiments, wherein the cells are to beadministered for therapy in a certain dosage, the amount of time betweeninception of culture of the cells and administration of a certain doseof cells is reduced (e.g., the time to reach a certain population isreduced as compared to culture methods employing non-physiologicconcentrations of oxygen.

In several embodiments, culturing stem cells in physiologic oxygenconcentrations (and/or in antioxidant compositions) positively affectsthe cells even after the period of culturing is complete (e.g., thecells have been administered to a subject). For example, in someembodiments, the use of physiologic oxygen concentrations imparts tostem cells a greater viability in vivo. As such, cells culturedaccording to the methods and/or with the use of compositions asdisclosed herein, remain viable for a longer period of timepost-administration, thereby increasing the potential effectiveness ofthese cells in cellular therapies. In several embodiments, cellscultured according to the methods and/or with the use of compositions asdisclosed herein engraft into host tissue to a greater degree than thosecultured in non-physiologic concentrations of oxygen. In certainembodiments, engraftment is a threshold step to efficacious celltherapy. For example, in some therapies, direct tissue regenerationplays a significant role in the therapy. However, advantageously, insome embodiments, cells cultured according to the methods and/or withthe use of compositions as disclosed herein generate a more effectiveamount of certain cellular signaling factors (e.g., autocrine,paracrine, intracrine or endocrine factors such as growth factors,hormones, cytokines and the like) that invoke an indirect mechanism oftherapy. For example, in some embodiments, an indirect mechanism, suchas recruitment of other endogenous cells is induced by a signalingfactor. As such, in some embodiments, a greater efficacy of therapy isachieved by virtue of the production (or reduced production) of certainsuch factors. In some embodiments, these signaling factors act inconcert with direct mechanisms to achieve an effective therapy, while insome embodiments, the signaling factors function alone. In severalembodiments, these direct and/or indirect mechanisms yield a moreeffective therapy, either by improving anatomical aspects of the targettissue, functional aspects of the target tissue, or combinationsthereof. For example, in the context of cardiac stem cell therapy,culture of cardiac stem cells in physiologic oxygen concentrationsimproves the viability and engraftment of administered cells, asdiscussed below. Moreover, these improved parameters, in the context ofa cell therapy for treating an adverse cardiac event, also yieldimproved cardiac anatomy (e.g., reduced infarct size, lower leftventricular area) and improved function (increased left ventricularejection fraction, increased cardiac output, etc.).

In some embodiments, the stem cells are embryonic stem cells. In someembodiments, the stem cells are adult stem cells. In some embodiments,the stem cells are hematopoietic stem cells, neuronal stem cells,mesenchymal stem cells, insulin producing stem cells, hepatocyte stemcells, or epithelial stem cells, or combinations thereof. In someembodiments, the stem cells comprise cardiac stem cells. In some suchembodiments, the cardiac stem cells are cardiospheres orcardiosphere-derived cells (CDCs). In some embodiments, the stem cellsare processed and prepared for administration to a subject in order torepair damaged tissue. In some embodiments, the subject has damagedcardiac tissue in need of repair. In some embodiments, the subject is amammal. In some embodiments, the subject is human, while in someembodiments the subject is a non-human mammal or other organism. Damagedcardiac tissue in a subject may be caused by a variety of events,including, but not limited to myocardial infarction, ischemic cardiactissue damage, congestive heart failure, aneurysm,atherosclerosis-induced events, cerebrovascular accident (stroke), andcoronary artery disease.

In one embodiment of the invention, cardiospheres and CDCs are isolatedas according to the following general protocol. Briefly, cardiac tissuesamples are weighed, cut into small fragments and cleaned of grossconnective tissue, and washed in a sterile solution, such asphosphate-buffered saline. In some embodiments, the tissue fragments areat least partially digested with protease enzymes such as collagenase,trypsin, and the like. In certain embodiments, the digested pieces areplaced in primary culture as explants on sterile tissue culture disheswith a suitable culture media. The digested pieces of tissue range insize from about 0.1 mm to about 2.5 mm (e.g., about 0.1-0.5 mm, 0.5-1mm, 1-2 mm, 2-2.5 mm, and overlapping ranges thereof). In severalembodiments, the digested pieces of tissue range 0.25 mm to about 1.5mm. Smaller or larger pieces of tissue can be used in other embodiments.The tissue culture dish and culture media are selected so that thetissue fragments adhere to the tissue culture plates. In someembodiments, the tissue culture plates are coated with fibronectin orother extracellular matrix (ECM) proteins, such as collagen, elastin,gelatin and laminin, for example. In other embodiments, the tissueculture plates are treated with plasma. In certain embodiments, thedishes are coated with fibronectin at a final concentration of fromabout 10 to about 50 μg/mL. In still other embodiments, the fibronectindishes are coated with fibronectin at a final concentration of fromabout 20 to 40 μg/mL, with still other embodiments employing a finalfibronectin concentration of about 25 μg/mL.

In certain embodiments, the base component of the complete explantmedium comprises Iscove's Modified Dulbecco's Medium (IMDM). In someembodiments, the culture media is supplemented with fetal calf serum(FCS) or fetal bovine serum (FBS). In certain embodiments, the media issupplemented with serum ranging from 5 to 30% v/v. In other embodiments,the culture media is serum-free and is instead supplemented withspecific growth factors or hydrolyzed plant extracts. In otherembodiments, the media is further supplemented with antibiotics,essential amino acids, reducing agents, or combinations thereof. In oneembodiment, the complete explant medium comprises IMDM supplemented withabout 20% fetal bovine serum, about 50 μg/mL gentamicin, about 2 mML-glutamine, and about 0.1 mM 2-mercaptoethanol. In some embodiments,the explant media is changed every 2-4 days while the explants culture.

The tissue explants are cultured until a layer of stromal-like cellsarise from adherent explants. This phase of culturing is furtheridentifiable by small, round, phase-bright cells that migrate over thestromal-cells. In certain embodiments, the explants are cultured untilthe stromal-like cells grow to confluence. At or before that stage, thephase-bright cells are harvested. In certain embodiments, phase-brightcells are harvested by manual methods, while in others, enzymaticdigestion, for example trypsin, is used. The phase-bright cells may betermed cardiosphere-forming cells, and the two phrases are usedinterchangeably herein.

Cardiosphere-forming cells may then be seeded on sterile dishes andcultured in cardiosphere media. In certain embodiments, the dishes arecoated with poly-D-lysine, or another suitable natural or syntheticmolecule to deter cell attachment to the dish surface. In otherembodiments, for example, laminin, fibronectin, poly-L-orinthine, orcombinations thereof may be used.

In certain embodiments, the base component of the cardiosphere mediumcomprises Iscove's Modified Dulbecco's Medium (IMDM). In someembodiments, the culture media is supplemented with fetal calf serum(FCS) or fetal bovine serum (FBS). In certain embodiments, the media issupplemented with serum ranging from 5 to 30% v/v, including from about5% to about 10%, about 10% to about 15%, about 15% to about 20%, about20% to about 25%, about 25% to about 30%, and overlapping rangesthereof. In other embodiments, the culture media is serum-free and isinstead supplemented with specific growth factors or hydrolyzed plantextracts. In certain other embodiments, the media is furthersupplemented with antibiotics, essential amino acids, reducing agents,or combinations thereof. In one embodiment the cardiosphere mediumcomprises IMDM supplemented with about 10% fetal bovine serum, about 50μg/mL gentamicin, about 2 mM L-glutamine, and about 0.1 mM2-mercaptoethanol.

According to one embodiment, cardiospheres will form spontaneouslyduring the culturing of the cardiosphere forming cells. Cardiospheresare recognizable as spherical multicellular clusters in the culturemedium. Cells that remain adherent to the poly-D-lysine-coated dishesare discarded. In certain embodiments, the cardiospheres are collectedand used to seed a biomaterial or synthetic graft. In other embodiments,the cardiospheres are further cultured on coated cell culture flasks incardiosphere-derived stem cell (CDC) medium.

In some embodiments used to culture cardiospheres into CDCs, theculturing flasks are fibronectin coated, though in other embodimentsother cellular attachment promoting coatings are employed. The culturedcardiospheres attach to the surface of the flask and are expanded as amonolayer of CDCs. CDC medium comprises IMDM, and in certain embodimentsis supplemented with fetal calf serum (FCS) or fetal bovine serum (FBS).In some embodiments, the media is supplemented with serum ranging from 5to 30% v/v. In other embodiments, the culture media is serum-free and isinstead supplemented with specific growth factors or hydrolyzed plantextracts. In certain other embodiments, the media is furthersupplemented with antibiotics, essential amino acids, reducing agents,or combinations thereof. In one embodiment, the CDC medium comprisesIMDM supplemented with about 10% fetal bovine serum, about 2 mML-glutamine, and about 0.1 mM 2-mercaptoethanol. CDCs may be repeatedlypassaged by standard cell culture techniques and in several embodimentsare harvested and used to seed a biomaterial or synthetic graft.

In some embodiments the cells to be administered to the subject areobtained from healthy tissue of the subject, e.g., an autologoustransplant. In some embodiments the cells to be administered to thesubject are obtained from healthy tissue of an individual other than thesubject, e.g., an allogeneic transplant. In some embodiments the cellsto be administered to the subject are obtained from healthy tissue anindividual who is highly genetically similar or identical to thesubject, e.g., a syngeneic transplant. In still further embodiments, thecells to be administered to the subject are obtained from healthy tissuean individual of a species distinct from the subject, e.g., a xenogeneictransplant.

In some embodiments, the culture methods reduce the concentration of ROSduring the cell culturing process. As used herein, the terms reactiveoxygen species or ROS shall be given their ordinary meaning and shallinclude, but not be limited to, superoxide anions (O₂—), hydrogenperoxide (H₂O₂), hydroxyl radicals (OH—), organic hydroperoxides (ROOH),alkoxy (RO) and peroxy (ROO) radicals, hypochlorous acid (HOCl), andperoxynitrite (ONOO—), and combinations thereof.

In several embodiments, antioxidant compositions are provided in a rangeof about 0.1 to about 1000 μM. In some embodiments, about 0.3 to about50 μM of a custom antioxidant cocktail (e.g., a formulation comprisingvitamin C, vitamin E, and glutathione) reduces the formation of ROS. Insome embodiments, ROS production is reduced by about 10-30%. In someembodiments ROS production is reduced by about 20-50%, or at least about25, 28, 31, 34, 37, 40, 43, 46, and 49%. In some embodiments, ROSproduction is reduced by about 40-80%. In some embodiments, ROSproduction is reduced by up to about 90%. As discussed below, in someembodiments, cells with a large reduction in ROS still exhibit DNAdamage. In some embodiments, use of the antioxidants described hereinreduce the formation, viability, and/or activity of ROS, and/or enhancethe degradation of ROS. In some embodiments, an antioxidant cocktail (orcomposition) comprises one or more of the following antioxidants:vitamin A, vitamin C, vitamin E, glutathione, mixed carotenoids (e.g.,beta carotene, alpha carotene, gamma carotene, lutein, lycopene,phytopene, phytofluene, and astaxanthin), selenium, Coenzyme Q10,indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechins,salicylic acid, curcumin, bilirubin. oxalic acid, phytic acid, lipoicacid, vanilic acid, polyphenols, flavanoids, ferulic acid, theaflavins,derivatives thereof, and other antioxidants.

In some embodiments, in addition to or in lieu of the decreasedproduction of ROS, the scavenging (e.g., processing) of ROS isincreased. In some embodiments, the activity of ROS scavenging enzymesis increased. In some embodiments, superoxide dismutase expressionand/or function is increased. In some embodiments, glutathioneperoxidase expression and/or function is increased. In still furtherembodiments, one or more of lactoperoxidase, catalase, andperoxiredoxins expression and/or function is increased.

In some embodiments, the culture methods reduce the mitochondrialmetabolism and therefore reduce the formation of ROS and/or the leakageof ROS from the mitochondrial membrane. In some embodimentsintracellular concentration of adenosine triphosphate (ATP) is reduced,either alone or in combination with a decrease in mitochondrial membranepotential. However, in other embodiments, the incidence of karyotypicabnormalities is reduced in the absence of significant changes in ATPconcentrations or mitochondrial membrane potential.

In several embodiments, the culture methods described herein decreasethe DNA damage caused by ROS formation. In some embodiments, one or moremarkers of DNA damage is reduced, as compared to stem cells culturedunder standard conditions and/or with standard media. In someembodiments, γ-H₂AX mRNA and/or protein expression is decreased when acertain concentration range of antioxidant cocktail is employed, asdiscussed in more detail below. Numerous other markers for various typesof DNA or histone damage that are known in the art may also be used toevaluate the efficacy of the methods and compositions disclosed herein.

As discussed above, some tissues have lower in vivo oxygenconcentrations. Thus, in some embodiments, the culturing methodcomprises reducing the oxygen concentration in which the cells arecultured. In some embodiments, in vitro O₂ concentrations that aresubstantially equivalent to in vivo O₂ concentrations are used. In someembodiments, O₂ concentrations less than 20% are used. In someembodiments, O₂ concentrations of about 10-15% are used, while in someembodiments, O₂ concentrations of about 13-19% are used. In otherembodiments, O₂ concentrations of less about 10% are used, including 9%,8%, 7%, and 6%. In still other embodiments, O₂ concentrations of about5% or less are used, including 4%, 3%, 2%, and 1%. In one embodiment,the closer the culture conditions in which a stem cell is culture are toits native oxygen conditions, the fewer karyotypic abnormalities willmanifest, as the cells is being cultured in what is effectively a morenatural environment. In contrast, non-physiological hyperoxic conditionsin culture may lead to oxidative stress, which, as discussed above, mayinduce ROS formation. In other embodiments, karyotypic abnormalities arereduced when O₂ concentrations are about 5-75% lower in vitro, ascompared to the in vivo environment.

In several embodiments, the culture method comprises supplementation ofculture media with an antioxidant cocktail. Several embodiments of thecomposition of the cocktail are discussed in more detail below. In someembodiments, the addition of antioxidants increases the consumption(e.g., scavenging) of ROS. In turn, the reduction in ROS reduces theamount of DNA damage. In several embodiments, the addition ofantioxidants increases the innate DNA repair mechanisms already presentin the cell. In some embodiments, the function of the existing DNArepair enzymes is upregulated, while in some embodiments, the expressionof one or more components of one or more DNA repair mechanisms isincreased.

Despite the negative effects of excessive ROS formation, excessivereduction in ROS levels can also be damaging to cultured cells. In oneembodiment, significant reductions in ROS levels may cause adownregulation of the innate DNA repair mechanisms (either expression,function, or both). Though unexpected, excessive reduction of ROSlevels, as could be achieved by certain levels of antioxidantsupplementation, could induce DNA damage due to the lack of sufficientDNA repair mechanisms or activity.

Advantageously, several embodiments of the compositions and culturemethods disclosed herein strike an optimal balance between ROS reductionand maintenance of adequate levels of innate DNA repair. In other words,in some embodiments, the culture methods and compositions used hereindecrease ROS levels sufficient to reduce DNA damage due to oxidativestresses, but at the same time do not reduce ROS levels to the degreethat innate DNA repair function is compromised. In several embodiments,antioxidant compositions are added in a range of about 0.1 to about 1000μM. While both high and low concentrations of antioxidants appear toresult in high levels of DNA damage, intermediate concentrationsunexpectedly result in reduced levels of DNA damage. In someembodiments, DNA damage is reduced when an antioxidant cocktail is addedin a concentration of about 0.1 to about 200 μM (e.g., about 0.1-20 μM,1-20 μM, 5-20 μM, 10-20 μM, 10-30 μM, 0.1-50 μM, 1-50 μM, 10-50 μM,25-50 μM, and overlapping ranges thereof). In some embodiments, theantioxidant cocktail is added in a concentration of about 5 μM to about30 μM. In some embodiments, about 10 μM is used. In some embodiments,about 20 μM is used.

In several embodiments, compositions comprising various antioxidantcompounds are provided in order to decrease the incidence of karyotypicabnormalities in the cultured cells, e.g., as disclosed above.Compositions disclosed herein may be used to affect any of the methodsdisclosed herein, unless otherwise specified. In some embodiments, thecompositions are used to supplement a culture media.

In several embodiments, the composition comprises one or morenon-peptide antioxidants. As used herein, the term “non-peptideantioxidant” shall be given its ordinary meaning and shall also beunderstood to include vitamins and other chemical compounds capable ofreducing ROS molecules, such as thiols (e.g., mercaptans) orpolyphenols. In some embodiments, the composition comprises one or moreantioxidant vitamins, including, but not limited to, vitamin A, vitaminC (ascorbic acid), and/or vitamin E. In some embodiments, one or more ofvitamin A, C or E are provided at a concentration ranging from about 0.1to about 1000 μM. In some embodiments, one or more of Vitamin A, C, or Eare added in a concentration ranging from about 1 to about 500 μM. Insome embodiments, one or more of Vitamin A, C, or E are added in aconcentration ranging from about 0.1 to about 200 μM (e.g., about 0.1-20μM, 1-20 μM, 5-20 μM, 10-20 μM, 10-30 μM, 0.1-50 μM, 1-50 μM, 10-50 μM,25-50 μM, 50-100 μM, 100-200 μM, and overlapping ranges thereof). Insome embodiments, one or more of Vitamin A, C, or E are added in aconcentration ranging from about 75 to about 150 μM, including about 80,90, 100, 110, 120, 130, and 140 μM. In some embodiments, one or more ofVitamin A, C, or E are added in a concentration ranging from about 1 toabout 50 μM, including about 5, 10, 20, 30, and 40 μM. Other non-peptideantioxidants include, but are not limited to: mixed carotenoids (e.g.,beta carotene, alpha carotene, gamma carotene, lutein, lycopene,phytopene, phytofluene, and astaxanthin), selenium, Coenzyme Q10,indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechins,theaflavins, salicylic acid, curcumin, bilirubin. oxalic acid, phyticacid, lipoic acid, vanilic acid, polyphenols, penicillamine, flavanoids,ferulic acid, theaflavins, derivatives thereof, and other antioxidants.In several embodiments, such non-peptide antioxidants are used in theconcentrations described above.

In several embodiments, the composition comprises one or more peptideantioxidants. As used herein, the term “peptide antioxidant” shall begiven its ordinary meaning and shall also be understood to includefull-length proteins, protein fragments, enzymes or polypeptides, aminoacids, or poylpeptide precursor molecules. In some embodiments, thepeptide antioxidant is added in addition to the non-peptide antioxidantdescribed above. In other embodiments, the peptide antioxidant (or thenon-peptide antioxidant) is used alone to supplement the culture media.In some embodiments polypeptides such as glutathione are incorporatedinto the composition. In some embodiments, glutathione is incorporatedinto the composition at a concentration ranging from about 0.1 to about1000 μM. In some embodiments, glutathione is added in a concentrationranging from about 1 to about 500 μM. In some embodiments, glutathioneis added in a concentration ranging from about 0.1 to about 200 μM(e.g., about 0.1-20 μM, 1-20 μM, 5-20 μM, 10-20 μM, 10-30 μM, 0.1-50 μM,1-50 μM, 10-50 μM, 25-50 μM, 50-100 μM, 100-200 μM, and overlappingranges thereof). In some embodiments, glutathione is added in aconcentration ranging from about 75 to about 150 μM, including about 80,90, 100, 110, 120, 130, and 140 μM. In some embodiments, glutathione isadded in a concentration ranging from about 1 to about 50 μM, includingabout 5, 10, 20, 30, and 40 μM. Other peptide antioxidants include, butare not limited to: superoxide dismutases, peroxiredoxins, thioredoxin,ceruloplasmin, transferrins, hydroperoxide reductases, n-acetylcysteineand, in several embodiments, are used in the concentrations describedabove.

In several embodiments, methods for assessing a subject's risk ofdeveloping a neoplastic disease are provided. In one embodiment, themethod comprises obtaining and evaluating a tissue sample from thesubject for evidence of karyotypic abnormalities (e.g., DNA damage) andcorrelating any detected abnormalities with the subject's antioxidantlevels. Thereafter, the subject is optionally treated to reduce orincrease antioxidant levels as needed. In several embodiments, DNAdamage is detected by analysis of markers of DNA damage, quantifying DNAdamage-associated mRNA expression levels, and/or quantifying DNAdamage-associated protein expression levels. In one embodiment, the riskof developing neoplastic disease increases as the incidence of DNAdamage increases, as the expression of DNA damage-associated mRNAincreases, and/or as the expression of DNA damage-associated proteinincreases. In some embodiments, identification of γ-H₂AX foci,quantifying γ-H₂AX mRNA expression levels, and/or by quantifying γ-H₂AXprotein expression levels, or combinations thereof, are used to detectand/or quantify DNA damage.

In several embodiments, a method for optimizing a subject's intake of anantioxidant-containing supplement is provided. In one embodiment, themethod comprises measuring the concentration of one or more reactiveoxygen species in a sample obtained from the subject (e.g., a tissue orblood sample) and measuring the incidence of DNA damage in cellsisolated from said sample. In some embodiments, the expression of DNArepair enzymes in cells isolated from said sample is measured. In someembodiments, both DNA damage and expression of DNA repair enzymes aremeasured.

In one embodiment, the method further comprises administration of anantioxidant composition to the subject (and optionally adjusting saidcomposition in subsequent administrations) in order to achieve anoptimal degree of homeostasis between the concentration of reactiveoxygen species in the subject and the subject's endogenous DNA repairmechanism(s). Such a level of reactive oxygen species can be assessedby, for example, expression of DNA repair enzymes or other markers ofDNA repair. In several embodiments, antioxidant compositions (e.g.,supplements) comprising an optimal amount of antioxidant-containingsupplement are provided. Such compositions are formulated to reduce DNAdamage caused by oxidative-stress but to minimize negatively impacting(wholly or partially) the subject's endogenous DNA repair mechanisms. Inseveral embodiments, the methods comprise serial administrations ofantioxidant compositions having different concentrations of antioxidantcompounds to a subject. In some embodiments, the invention comprisesoptimizing antioxidant therapy for an individual. In one embodiment, afirst sample (e.g., tissue or blood sample) is obtained from the subjectprior to administration, in order to establish a baseline. Thereafter, afirst antioxidant composition having a first concentration of anantioxidant composition is administered. An additional sample isobtained from the subject and evaluated for concentrations of reactiveoxygen species, markers of DNA damage, changes in expression of DNArepair enzymes, or combinations thereof. In some embodiments, aplurality of additional administrations of an antioxidant composition(optionally having varying concentrations of antioxidant compounds) maybe made in conjunctions with obtaining and evaluating a plurality ofadditional samples from the subject. As such, an optimal concentrationof antioxidant compounds may be reached over time by the serialevaluation of the subject's response to the antioxidant compositions.Thus, in several embodiments, therapeutic antioxidant compositions thatbalance oxidative damage with endogenous DNA repair mechanisms areprovided based on a subject's individual antioxidant and/or karyotypicprofile. Antioxidant compositions according to several embodimentsherein comprise, consist, or consist essentially of one, two, three,five, ten, or more antioxidants.

EXAMPLES

Examples provided below are intended to be non-limiting embodiments ofthe invention.

Materials and Methods

The following materials, methods, and protocols may be used to performthe examples disclosed herein as well as practice the variousembodiments of the invention disclosed herein.

Long-Term Culture Conditions for Human CDCs

Adult human cardiac stem cells were isolated from percutaneous septalendomyocardial heart tissue biopsies (about 10-20 μg), which wereobtained from patients during clinically-indicated procedures (tomonitor heart transplant recipients for rejection) after informedconsent. Biopsies were minced into small fragments and digested with 0.2mg/ml collagenase for 30 minutes. The digested tissue fragments werethen equally moved to each of four 6-cm diameter culture dishes coatedwith 20 μg/ml fibronectin (BD Biosciences), and randomly selected toculture as “explants” in the following four conditions:

-   -   1) in a typical 20% O₂ incubator (95% air/5% CO₂);    -   2) in a 5% O₂ “hypoxia incubator”;    -   3) in a typical 20% O₂ incubator with the addition of 1000-fold        diluted proprietary antioxidant supplement (Sigma-Aldrich,        Catalogue Number: Sigma A1345, Antioxidant A);    -   or    -   4) a custom antioxidant cocktail consisting of 100 μM        L-ascorbate, L-glutathione, and alpha-tocopherol acetate        (Sigma-Aldrich, Antioxidant B).

The cardiosphere and CDC amplification steps were performed under thesame conditions in each group. IMDM basic medium (Gibco) supplementedwith 10% FBS (Hyclone) and 20 mg/ml gentamycin was used for allcultures. As used herein, the term “short-term culture” shall be givenits ordinary meaning and also be read to include culture periods rangingfrom about 1 to about 48 hours, including, 8, 16, 20, 24, 32, 36, and 40hours. As used herein, the term “long-term culture” shall be given itsordinary meaning and also be read to include culture periods rangingfrom 48 hours to 6 months. In some embodiments, long-term culture lastsfor several days to several months, including 2, 3, 4, and 5 months. Insome embodiments, long-term culture lasts for several days to severalweeks, include 1-3 weeks, 2-5, weeks, 3-7 weeks, and 5-10 weeks. In someembodiments long-term culture lasts for about 6 to about 10 months, andin some embodiments from about 10 months to several years.

Karyotype Analysis

Twice-passaged CDCs (with long-term culture for 1-2 months from the dateof tissue biopsy) were seeded onto fibronectin-coated 25-cm² tissueculture flask (10⁴ cells/cm²). After ˜24 hours of incubation, cells weretreated with 0.1 μg/ml colcemid (Invitrogen) for 4 hours, thentrypsinized, treated with hypotonic solution, and fixed. Metaphases werespread on microscope slides, and karyotype analysis was done by usingstandard G banding technique. The chromosomes were classified accordingto the International System for Human Cytogenetic Nomenclature. At least20 metaphases were analyzed per cell sample.

Determination of ROS Levels

To assay intracellular ROS levels (ROS levels), twice-passaged CDCs wereseeded in 6- or 96-well plates coated with 20 μg/ml fibronectin, andcontinuously cultured under the abovementioned four differentconditions. At about 90% confluence, cells were incubated with 10 μM2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen) for 60min to allow DCFH-DA to diffuse into cells. The DCF fluorescenceintensity in cells cultured in 96-well plates is directly determinedusing SpectraMax® M5 (Molecular Devices Corp.) with an excitationwavelength of 495 nm and an emission wavelength of 520 nm. Cellscultured in 6-well plates were trypsin-treated and fixed. The DCFfluorescence intensity in cells was analyzed using a FACS Calibur flowcytometer with CellQuest software (BD Biosciences).

To observe the changes of ROS levels during short-term exposure todifferent concentrations of antioxidants, catalase, and H₂O₂, CDCs wereexpanded by traditional conditions under 20% O₂ as described above.Twice-passaged cells were seeded in 6- or 96-well plates coated with 20μg/ml fibronectin. When about 70% confluent, cell cultures weresupplemented with antioxidant A (100-1,000,000-fold dilution), customantioxidant cocktail “B” (0.1-1000 μM), catalase (0.1-1000 units/ml), orH₂O₂ (0.1-1000 μM), as indicated. After 24 hours of culture, the DCFfluorescence intensity in cells was measured using the same methods asdisclosed above.

Analysis of DNA Damage in CDCs and ES Cells

DNA damage in human CDCs and ES cells was evaluated by immunostainingfor phosphorylation of histone H₂AX on serine 139 (γ-H₂AX), a marker ofDNA double-strand breaks, after short-term culture with differentconcentrations of antioxidants, catalase, and H₂O₂. CDCs were expandedin conventional conditions under 20% O₂ as described above.Twice-passaged CDCs and ES cells were used in these Examples. Cells wereseeded in 96-well plates coated with 20 μg/ml fibronectin. When about70% confluent, cell cultures were supplemented with antioxidant A(100-1,000,000-fold dilution), custom antioxidant cocktail “B” (0.1-1000μM), catalase (0.1-1000 units/ml), or H₂O₂ (0.1-1000 μM), as indicated.After 24 hours of culture, cells were fixed, permeabilized and stainedwith rabbit polyclonal antibody against γ-H₂AX (phosphor 5139, AbeamInc.). After being washed, the cells were stained with a PE-conjugatedsecondary antibody and 4,6-diamidino-2-phenylindole (DAPI).Quantification of cells positive for γ-H₂AX foci was performed byfluorescence microscopy (×40 magnification). Briefly, at least 5 imageswere captured from each culture condition from randomly-selected fieldsusing Q-imaging (RETIGA EXi FAST, Canada) with the same exposure time.Cells with γ-H₂AX foci in the nuclei were counted by a single observerblinded to treatment regimen, and the percentage of cells with γ-H₂AXfoci in each culture condition was used for statistical analysis.

To quantify DNA damage in long-term cultured CDCs under theabovementioned four different conditions, twice-passaged CDCs wereseeded in 6- or 96-well plates coated with 20 μg/ml fibronectin, andcontinuously cultured for 24 hours. The analysis of γ-H₂AX foci was doneas described above.

Western Blotting

To examine the protein levels of ATM and other DNA repair-relatedfactors, total protein was purified from twice-passaged CDCs culturedunder the abovementioned four different conditions, usingwell-established laboratory techniques. Briefly, harvested cells werehomogenized in a lysis buffer containing a protease inhibitor mixture(Roche Applied Science) on ice. After centrifugation at 15,000 rpm for10 min, the supernatant was collected for experiments. The equivalent of30 μg of total protein was loaded onto 5% or 10% SDS-PAGE gels, and thentransferred to PVDF membranes. After overnight blocking in 3% milkTBS-T, membranes were incubated with the following primary antibodies:1:5000 dilution of rabbit anti-ATM polyclonal antibody, 1:1000 dilutionof rabbit anti-ATR polyclonal antibody, 1:500 dilution of rabbitanti-Chk1 (phosphor S317) polyclonal antibody, 1:200 dilution of rabbitanti-Chk2 (phosphor T26) polyclonal antibody, 1:1000 dilution of mouseanti-Rad50 monoclonal antibody, 1:1000 dilution of mouse anti-Rad51monoclonal antibody (all from Abeam Inc.) and 1:3000 dilution of rabbitanti-β-actin monoclonal antibody. The appropriate horseradishperoxidase-conjugated secondary antibodies were used, and then the blotswere visualized by using SuperSignal West Femto maximum sensitivitysubstrate (Thermo Scientific)) and exposed to Gel Doc™ XR System(Bio-Rad Lab., Inc.). Quantitation for blots was done by Quantity Onesoftware, and expressions were normalized by β-actin.

To observe the expression of ATM and other DNA repair-related factorsduring short-term exposure to different concentrations of antioxidants,catalase, and H₂O₂, CDCs were expanded by traditional culture in 20% O₂as described above. Twice-passaged cells were seeded in 6-well platescoated with 20 μg/ml fibronectin. When about 70% confluent, cells werecultured in 20% O₂ with the supplement of antioxidant A(100-1,000,000-fold dilution), custom antioxidant cocktail “B” (0.1-1000μM), catalase (0.1-1000 units/ml), or H₂O₂ (0.1-1000 μM), as indicated.After 24 hours of culture, cells were harvested and total protein waspurified. The expression of ATM and other DNA repair-related factors wasassessed by Western blotting as described above.

Measurement of the Intracellular ATP Level and MitochondrialTransmembrane Potential

The intracellular ATP level was measured by the luciferin-luciferasemethod using an ATP-determination kit (Invitrogen). Briefly,twice-passaged CDCs (2×10⁵ cells/well) were seeded in 6-well platescoated with 20 μg/ml fibronectin, and continuously cultured under theabovementioned four different conditions for 24 hours. The cells werewashed twice with ice-cold PBS and lysed in 200 μl lysis buffer withprotease inhibitors. The lysates (20 μl normalized by protein content)were added to the reaction solution (200 μl) containing 0.5 μMluciferin, 1.25 μg/ml luciferase, and 1 μM DTT, and the bioluminescencewas measured using a Monolight™ 3010 (Pharmingen).

To measure mitochondrial transmembrane potential, twice-passaged CDCs(2×10⁵ cells/well) were seeded in 6-well plates coated with 20 μg/mlfibronectin, and continuously cultured under the abovementioned fourdifferent conditions for 24 hours. Cells were loaded with TMRE at 37° C.for 30 minutes, and harvested by trypsinization. The fluorescenceintensity in cells was analyzed using a FACS Calibur flow cytometer withCellQuest software (BD Biosciences).

Statistical Analysis

All results are presented as mean±SD. Statistical significance wasdetermined using the 2-tailed chi-square test for karyotype data andANOVA followed by Bonferroni post hoc test for other data (Dr. SPSS II).Differences were considered statistically significant when p<0.05.

Example 1 Genomic Alterations Decrease in Physiological Oxygen butUnexpectedly Increase with Antioxidant Supplements

Source human heart biopsies (n=16) were divided and processed inparallel in the various culture conditions, facilitating directcomparisons. CDCs grown under conventional conditions not infrequentlyincluded cells with genomic alterations (6 of 16 samples; FIG. 1, Table1 and Table 2). In reference to FIG. 1, each bar represents a histogramof one sample of stem cells; blue denotes cells with a normal karyotype.Compared with culture in traditional 20% O₂ incubator (95% room air/5%CO₂), the number of cells with DNA breaks or translocations (coloredgreen) and losses or gains of chromosomes (red) was decreased when cellswere cultured in 5% O₂ (p=0.007). When CDCs were cultured in 5% O₂,genomic alterations were detected in only 3 of 16 samples (FIG. 1, Table2). The genomic changes were relatively innocuous: one sample containedone cell with a balanced translocation, another had 8 cells with aderivative chromosome, and the third included one cell with loss of theY chromosome (FIG. 1, Table 2). The reduction of the frequency ofchromosomal abnormalities in CDCs cultured in 5% O₂ indicates thatphysiological oxygen concentrations during culture enhance the genomicstability of stem cells.

TABLE 2 Karyotyping Date of Twice-Passaged Human CDCs Samples 20% O₂Antioxidant A Antioxidant B 5% O₂ CSB59m 46, XY[20] 46, XY[20] 46,XY[20] 46, XY[20] CSB59s 46, XY[20] 45, X − Y[10]/46, XY[10] 46, XY[20]46, XY[20] CSB61m 46, XY[20] 47, XY, +7[3]/46, XY[17] 47, XY, +7[3]/46,XY[18] 46, XY[20] CSB61s 46, XY[20] 47, XY, +7[1]/46, XY[19] 46, XY[20]46, XY[20] CSB64m 46, XY[20] 48, XY, +2, +20[4]/46, XY, 46, XY[20] 46,XY[20] der(13)t(9; 13)(q12; p11, 2)[1]/46, XY[15] CSB64s 46, XY[20] 46,XY[20] 46, XY[20] 46, XY[20] CSB65m 47, XY, +8[9]/48, s1, +2, −9, 46,XY[20] 46, X, +8[7]/47, XY, +12 46, XY[20] der(13)t(9; 13)(q12; p11.1),[10]/46, XY[3] +14[1]/46, XY[11] CSB65s 45, X, −Y[2]/47, XY, 47,XYY[4]/46, XY[16] 47, XYY[11]/47, XY, +2 46, XY[20] +8[1]/46, XY[20][1]/47, XY, +8[5]/46, XY[3] CSB66m 47, XX, +2[1]/ 46, XX[20] 46, XX 46,XX[20] 47, XX, m[1]/46, XX[18] CSB66s 46, XX[20] 47, XX, +8[1]/46,XX[19] 46, XX, add(1)(p36.1)[3]/ 46, XX[20] 46, XX[18] CSB69m 47, XY,+8[2]/45, X, −Y 47, XY, +8[5]/46, XY[15] 47, XY, +8[4]/ 46, XY,der(18)t(14; 18) [1]/46, XY[18] 46, XY[16] (q22; q23)[3]/46, XY[17]CSB69s 46, XY[20] 47, XY, +8[17]/46, XY[3] 47, XY, +8[5]/ 46, XY,der(18)t(14; 18) 46, XY[15] (q22; q23)[5]/45, X, −Y [1]/46, XY[15]CSB73m 46, XY[20] ND ND 46, XY, t(6; 19)[1]/ 46, XY[19] CSB73s 46,XY[20] ND ND 46, XY[20] CSB76m 45, X, −Y[6]/46, XY[14] ND ND 46, XY[20]CSB76s 45, X, −Y[1]/47, XY, ND ND 46, XY[20] +8[1]/46, XY[18]

In contrast, karyotypic abnormalities were dramatically increased infrequency and severity when CDCs were cultured in 20% O₂ with either oftwo antioxidant cocktails: a proprietary antioxidant supplement for cellculture (product A1345, Sigma-Aldrich, 1000-fold dilution; AntioxidantA), or a custom antioxidant mixture (L-ascorbate, L-glutathione, andα-Tocopherol acetate, each 100 μM; Antioxidant B) (FIG. 1, Table 2).Among the 12 samples of CDCs cultured with Antioxidant A or B, 8 and 6samples included 46 and 49 cells with genomic alterations, respectively(p<0.001 vs. 20% O₂ culture by chi-square test, FIG. 1).

Unlike conventional 20% O₂ culture, where trisomy 8 and loss of Ypredominated, the karyotypic abnormalities seen with antioxidants werenumerous and varied (trisomy 2, 7, 8, 12, 18, and 20). FIG. 2 depicts(A) CDCs cultured in a traditional 20% O₂ incubator (95% air/5% C O₂),(B) gain of chromosome 7 in the presence of Antioxidant A, and (C) gainsof chromosome 2 and 20 in the presence of Antioxidant B. To theApplicant's knowledge, some of these, namely trisomy 7 and trisomies 2and 20 have not been previously reported (FIG. 2).

The effects of antioxidants on genomic stability appear not to reflectgeneralized toxicity, as CDCs proliferated normally without obviousmorphologic abnormalities (FIG. 3). Panel A depicts twice-passaged CDCsgrown for 36 days in 20% O₂ without addition of antioxidants. Panel Bdepicts CDCs grown in the same O₂ concentration in media supplementedwith an antioxidant supplement at 1:1000 dilution. Panel C depicts CDCsgrown in the same O₂ concentration in media supplemented with a customantioxidant cocktail at 100 μM. Panel D indicates that that nosignificant differences in CDC proliferation were detected among thevarious culture conditions.

Furthermore, neither the intracellular ATP level nor mitochondrialtransmembrane potential showed any obvious differences among the fourvarious culture conditions (FIGS. 4A and B, respectively). Indeed, thesimilarities in cell proliferative activity and intracellular ATP levelsindicate that energy metabolism is not severely undermined in any of thelong-term culture conditions.

Example 2 Antioxidants Decrease Intracellular ROS Monotonically but DNADamage Shows a Biphasic Response in Stem Cells

CDCs were initially maintained in traditional 5% CO₂/20% O₂ culturecondition and then seeded into 96-well plates and cultured for 24 hoursunder experimental conditions. Intracellular ROS levels in CDCs exposedfor 24 hours to a wide range of antioxidant concentrations (A-B), withcatalase (a pure ROS scavenger, C) and hydrogen peroxide (H₂O₂, apowerful oxidant, D) as controls were measured. The results shown inFIG. 5 are means±Std. Dev. for six separate experiments using differenttwice-passaged CDCs. (a.u.: arbitrary units. * p<0.01, p<0.05 vs. thebaseline levels., represented by “0” on the x-axis). The results shownin FIG. 6 are representative histograms of the intracellular ROS dataobtained by flow cytometry

Catalase decreased, and H₂O₂ increased, ROS levels in a progressivedose-dependent manner (FIG. 5C-D, FIGS. 6C-D). Like catalase,antioxidants A and B both decreased ROS levels monotonically (FIG. 5A-B,FIG. 6A-B). At the concentrations used to culture CDCs for karyotypinganalysis (1000-fold dilution of antioxidant A and 100 antioxidant B,respectively), ROS levels was very low.

Likewise, ROS levels was depressed in CDCs sent for karyotyping analysisafter 1-2 months in 5% oxygen, and even more so in antioxidants,relative to routine culture conditions (FIG. 7). As shown in Panel A,the intracellular ROS was lower in cells cultured long-term under 5% O₂than under 20% O₂. Intracellular ROS was decreased to very low levels bythe addition of 1000-fold diluted antioxidant supplement (Antioxidant A)or 100 μM custom antioxidant cocktail (Antioxidant B), as shown in PanelB. Shown in Panel C are representative images of γ-H₂AX foci in CDCs(arrows) cultured for long-term under different conditions. As depictedin Panel D, compared to traditional 20% O₂ culture, quantitative datashowed that γ-H₂AX foci in CDCs was significantly decreased in 5% O₂culture, but increased by the supplement with antioxidants. * p<0.01 vs.other groups, † p<0.01 vs. 5% O₂ and 20% O₂. Thus, assuming that ROSlevels in CDCs cultured under 5% O₂ approximates the physiologicalintracellular concentration, long-term culture with antioxidantssuppresses ROS levels to sub-physiological levels (FIGS. 5, 6, and 7).

The concept of “reductive stress” (an extreme suppression of ROS) mayunderlie a form of cardiomyopathy due to protein aggregation. Toidentify whether an excessive decrease of ROS levels likewise inducesDNA damage and genomic instability, γ-H₂AX foci was quantified in CDCsand human ES cells (FIG. 8). γ-H₂AX is a marker of DNA double-strandbreaks and was detected in several CDCs whether cultured with 1:1000diluted antioxidant supplement (Panel A), with 100 μM custom antioxidantcocktail (Panel B), with 100 units/mL catalase (Panel C), or with 100 μMH₂O₂ (Panel D).

It was also demonstrated that oxidative stress induced by H₂O₂ increasedDNA damage dose-dependently (FIG. 9D). Here, however, the effects wereunexpectedly biphasic: the percentage of CDCs with γ-H₂AX foci wasdecreased at low antioxidant concentrations, but increased at higherdoses (FIG. 9A-B). A similar result was observed with increasingconcentrations of catalase (FIG. 9C). The biphasic response is notlimited to CDCs: human ES cells exhibited a similar pattern (red crosseswith dotted lines, FIG. 9A-C), although the overall percentages of EScells with γ-H₂AX foci were lower than in adult CDCs. Interestingly, thenumber of γ-H₂AX foci was minimal at modest concentrations ofantioxidants (10,000-fold dilution of antioxidant A, 10 μM ofantioxidant B, and 10 units/ml of catalase; FIG. 9A-C), the sameconcentrations that drive ROS levels to “physiological” levels. Theseresults motivate the concept of an “oxidative optimum”, a narrow rangeof ROS levels within which stem cells maintain optimal genomicstability.

This dose-response data correlated with the results of FIGS. 7C and 7Ddiscussed above, where γ-H₂AX foci were significantly decreased in 5% O₂culture, but increased by supplementation with 1000-fold dilutedantioxidant supplement (Antioxidant A) or 100 μM custom antioxidantcocktail (Antioxidant B), when compared to traditional 20% O₂ culture(FIG. 7C, D).

Example 3 Extreme Suppression of Intracellular ROS Down-Regulates ATMand Other DNA Repair Factors

The protein kinase ATM (ataxia-telangiectasia mutated), is believed toplay a role in DNA repair. Intracellular ROS enhances the expression ofATM, which phosphorylates a host of downstream targets in response toDNA double-strand breaks, inducing cell cycle arrest and inhibitingapoptosis. It is possible that excessive suppression of ROS levels mightdown-regulate ATM, thereby promoting genomic instability. ATM proteinlevels were indeed decreased at high concentrations of antioxidants Aand B (>1000-fold dilution, (>100 μM, respectively), or catalase (>100units/ml) (FIG. 10A-C). As shown in Panel A, the protein levels of ATMin CDCs were decreased at high doses (<1000-fold dilution), but not atlow doses (>10,000-fold dilution) of antioxidant supplements(Antioxidant A). Panel B shows that ATM protein in CDCs decreasesat >100 μM, but not at <10 μM, of custom antioxidant cocktail(Antioxidant B). Panel C shows that catalase also decreases ATM proteinlevel in CDCs at >100 units/ml, but not at <10 units/mL. Finally, PanelD shows that the expression of ATM in CDCs was slightly increased at ahigh dose (>100 μM) of H₂O₂. Quantitative data are means±standarddeviation for four separate experiments using different twice-passagedCDCs.

Various other DNA repair factors, including ATR and downstream factorsRad50, Rad51, Chk1, and Chk2, were also decreased by antioxidants (FIGS.11 and 12), at the same concentrations that suppressed ROS levelssignificantly. As shown in FIG. 11, Panel A (antioxidant supplement),Panel B (custom antioxidant cocktail), Panel C (catalase), theexpression levels of ATR, Rad50, Rad51, Chk-1, and Chk-2 were slightlyor obviously decreased by high concentrations of catalase orantioxidants. In contrast, as shown in Panel D, expression levels wereincreased by high-dose H₂O₂. FIG. 12 shows representative quantitativehistograms of the expression data from FIG. 11.

According to one embodiment of the invention, the expression levels ofATM and other DNA repair-related factors were down-regulated when ROSlevels were decreased to “sub-physiological” levels at very highconcentrations of antioxidants. In one embodiment, a low concentrationof antioxidants drives ROS levels to an optimal “physiological” range,which reduces oxidative stress-induced DNA damage without impairing theDNA repair system. In one embodiment, given that ATM protein levels fellwithin 24 hours of exposure to antioxidants, physiological ROS levelsconcentrations may stabilize ATM and other DNA repair-related proteinkinases, consistent with the notion that reductive stress inducesintracellular protein aggregation. According to one embodiment,transcriptional downregulation plays an additional or supplemental role.In one embodiment, while ATM and related DNA repair factors may underliethe genomic instability seen with high antioxidants, the levels of theseproteins do not change when CDCs are grown in certain conditions (FIG.13). According to one embodiment, as shown in FIG. 13A, as compared tocells cultured under traditional 20% O₂, ATM expression did notsignificantly change in cells cultured under 5% O₂, but wassignificantly decreased by the addition of 1000-fold diluted antioxidantsupplement (Antioxidant A) or 100 μM custom antioxidant cocktail(Antioxidant B). According to one embodiment, as shown in FIG. 13 B,expression of ATR, Rad50, Rad51, Chk1, and Chk2 did not significantlychange in cells cultured under 5% O₂. However, according to someembodiments, many of these factors were decreased in cells culturedunder traditional 20% O₂ with the addition of 1000-fold dilutedantioxidant supplement (Antioxidant A) or 100 μM custom antioxidantcocktail (Antioxidant B).

According to several embodiments, the invention comprises compositionsand methods to balance DNA injury during oxidative stress and faulty DNArepair in reductive stress.

According to several embodiments, a biphasic relationship betweenintracellular ROS levels and genomic stability in human cardiac and EScells is provided. In one embodiment, a modest ROS suppression byculture in physiological oxygen (5%) decreases karyotypic abnormalities,but profound ROS suppression by antioxidant supplements paradoxicallyenhances genomic alterations. FIG. 14 depicts schematically, onepossible model for such results. In one embodiment, oxidative stressinduces DNA damage, accounting for the high frequency of karyotypicabnormalities in 20% O₂ culture (FIG. 14, right panels). On the otherhand, excessive suppression of ROS to subphysiological levelsdown-regulates DNA repair pathways, thereby contributing to genomicinstability (FIG. 14, left panels). Thus, according to one embodiment,optimal “physiological” levels of ROS are provided for activation of DNArepair pathways to maintain genomic stability in stem cells (FIG. 14,center panels). In one embodiment, excessive inhibition of ROSproduction leads to defective DNA repair and, thereby, increasedfrequency of karyotypic abnormalities seen with high antioxidants. Inone embodiment, maintenance or enhancement of DNA repair pathways usingthe compositions disclosed herein contribute to the reduction in random(rather then systematic) chromosomal changes. In other embodiments,reduction of systematic chromosomal changes is achieved. In yet otherembodiments, reduction in both random and systematic chromosomal changesis achieved.

According to some embodiments of the invention, CDCs derived frompost-transplant patients are used. In one embodiment, such CDCs exhibitchromosomal abnormalities at a higher frequency thannon-immunosuppressed patients. Thus, in several embodiments, theinvention is particularly beneficial for reducing chromosomal damage incells obtained from subjects with suppressed immune systems or otherconditions that enhance karyotypic abnormalities.

Several embodiments of the invention are unexpected because freeradicals are popularly viewed as harmful by-products of cell metabolism,and antioxidant dietary supplements, even at high doses, are touted tobe beneficial in the prevention of cancer and a host of other diseases.In one embodiment, a balanced formulation comprising vitamin C, vitaminE, and glutathione prevents the extreme suppression of ROS by high-doseantioxidants which would otherwise increase DNA damage and genomicinstability associated with down-regulation of DNA repair-relatedprotein kinases. Several embodiments of the invention facilitate thedual role of ROS, in which a physiological level of ROS is aids ineffective DNA repair, but high ROS induces DNA damage. In oneembodiment, compositions or hypoxic conditions disclosed herein reducethe incidence of karyotypic abnormalities in cells by more than 10%,25%, 50%, 75%, or 95% as compared to control cells.

Example 4 Effect of Physiologic Oxygen Concentrations on Stem CellProliferation

In order to determine other effects of that culturing in physiologicoxygen has, CDCs were generated in duplicate from 13 clinical biopsysamples. One portion of the tissue was used to generate cells which werecultured in room oxygen, while another portion was cultured inphysiologic oxygen. The mean CDC yield for room oxygen cultures was77.1±80.3 million from a mean starting mass of 263±185 mg of tissue.This yield was achieved in a mean time of 35.1±6.4 days. During the sameaverage manufacturing time, mean CDC yield increased 3.2-fold forspecimens cultured in physiologic oxygen. Moreover, the amount ofstarting material decreased 6.1-fold. The per mg yield was therefore19.6-fold higher. To confirm these findings, parallel runs with the sameamount of human starting tissue (˜60 mg) were performed. Culture inphysiologic oxygen conditions produced over six times more CDCs thanroom oxygen conditions (see FIG. 15).

In several embodiments, physiological oxygen concentrations also reducethe amount of time needed in culture to generate a given number ofcells. Thus, in several embodiments, use of physiological oxygenconcentrations is particularly advantageous because such conditionsincrease the overall yield of CDCs. In some embodiments, reducedquantities of starting cardiac tissue are required. In some embodiments,this is advantageous because a given sample has a greater capacity toproduce CDCs, which increases the number of CDCs that are available fortherapy and/or banking.

As discussed above, CDCs have been shown to occasionally acquirecytogenetic abnormalities, most frequently aneuploidy, during culture.In still additional embodiments, physiological oxygen concentrationsalso reduce decrease the incidence of one or more of aneuploidy, DNAbreaks, base-pair mismatches, or translocations (see, e.g., FIGS. 1 and16). Therefore, in several embodiments CDCs are cultured in physiologicoxygen to reduce cytogenetic abnormalities.

Example 5 In Vivo Efficacy of CDCs Grown in Physiologic Oxygen

As discussed above, culture conditions comprising physiological oxygenlevels are used in some embodiments. The in vivo efficacy of CDCscultured in physiologic oxygen was tested in comparison to CDCs culturedin room oxygen (20%). A mouse myocardial infarction model was used. Inbrief, myocardial infarction (MI) was created by ligation of themid-left anterior descending coronary artery. Cells were deliveredintramyocardially by direct injection at two peri-infarct sitesimmediately following ligation. CDCs (105) were injected in calcium-freePBS (5-7 μL at each site), with 10⁵ normal human dermal fibroblasts(NHDFs) or PBS as control. Cardiac function measured 3 weeks post-MI,indicated that CDCs grown in physiologic oxygen were yielded improvedleft ventricular ejection fraction (LVEF) as compared to CDCs grown inroom oxygen (see FIG. 17). Both CDC-treated groups outperformed the PBSgroup. These data indicate that CDC potency is enhanced when culture isperformed under physiologic oxygen conditions, as is done in severalembodiments. In several embodiments, the potency of CDCs cultured inphysiologic oxygen is increased in a statistically significant manner.In some embodiments, the potency of CDCs cultured in physiologic oxygenis increased by at least about 5%, about 10%, about 15%, about 20%,about 25% or greater.

Example 6 Expansion of Human Cardiac Stem Cells in Physiologic OxygenImproves Cell Production, Efficiency, and Potency for Myocardial Repair

Further to the discoveries detailed above, additional studies wereconducted to obtain additional details regarding the impact of culturinghuman cardiac stem cells in physiologic concentrations of oxygen.

Expansion of cells in culture is the foundation for a wide variety ofapplications of adult stem cells and embryonic stem (ES) cells,including regenerative medicine and drug development. Chromosomalabnormalities are frequently found in stem cells after long-term cultureand such chromosomal abnormalities may enhance carcinogenesis and impairfunctional potency, thereby complicating the therapeutic application ofstem cells. Therefore, it is important to expand stem cells with fewerchromosomal abnormalities for clinical applications. Resident cardiacstem cells are considered to be particularly promising for myocardialregeneration, as they mediate cardiogenesis and angiogenesis. In severalembodiments, the methods and compositions disclosed herein allow theexpansion of a relatively small number of cells into a larger population(e.g., tens of millions of cells in some embodiments) of cardiac stemcells and supporting cells (collectively termed CDCs) using ex vivoculture. As discussed above, in some embodiments, the initial sample ofcardiac tissue is from a minimally invasive human heart biopsy. In otherembodiments, other tissue sources are used (e.g., surgically obtainedcardiac tissue, donor cardiac tissue, etc.). While clinical applicationof CDCs is presently under way in the CADUCEUS (CArdiosphere-DerivedaUtologous Stem CElls to Reverse ventricUlar dySfunction,ClinicalTrials.gov. Identifier NCT00893360) trial, karyotyping revealedthat approximately one-third of preliminary CDC production runs includedcytogenetically abnormal cells, most often due to changes in chromosomenumber (aneuploidy; see e.g., FIG. 1).

As discussed above, ex vivo expansion of stem cells, including CDCs, isgenerally performed by incubating cells in incubators equilibrated with95% air and 5% CO₂ (˜20% O₂). However, the oxygen concentration of thein vivo microenvironment of stem cells in biological tissues is oftenmuch lower, ranging from about 1% to about 8%, (depending on thetissue). As a result, stem cells for use in a tissue having a low oxygenmicroenvironment in vivo may suffer from oxidative stress under suchconventional culture conditions. Given that oxidative stress may play arole in DNA damage and genomic instability, the present studyinvestigated the effects of ex vivo expansion of human cardiac stemcells in ‘hypoxic’ conditions (mimicking the low oxygen tensionsoperative in vivo)

Ex Vivo Expansion of Human Cardiac Stem Cells Under Low Oxygen

Adult human cardiac stem cells were expanded using similar methods asdescribed above. Briefly, endomyocardial heart tissue biopsies (˜10 mg)were obtained from patients during clinically indicated procedures afterinformed consent, in an Institutional Review Board-approved protocol. Asdiscussed above, in some embodiments, non-biopsy tissue sources areused. All investigations conform to the Declaration of Helsinki.Biopsies were minced into small fragments and digested with 0.2 mg/mL ofcollagenase for 30 min. The digested tissue fragments were then equallydivided and moved to two 6 cm diameter culture dishes coated with 20mg/mL of fibronectin (BD Biosciences) and randomly selected to cultureas ‘explants’ in a typical CO₂ incubator (20% O₂) or a incubator withphysiological low oxygen (5% O2). Following cardiosphere formation, CDCswere grown and expanded by passaging under 20% O₂ (20% O₂ CDCs) or 5% O₂(5% O₂ CDCs), respectively. IMDM basic medium (Gibco) supplemented with10% FBS (Hyclone) and 20 mg/mL gentamycin was used for all cultures.Twice-passaged CDCs (1-2 months culture from the date of tissue biopsy)were used for experiments unless otherwise indicated.

Karyotype Analysis

CDCs were seeded onto fibronectin-coated 25 cm² tissue culture flasks(10⁴ cells/cm²) and continuously cultured under 5% O₂ or 20% O₂,respectively. The karyotyping data in this example are extended fromthose discussed above. After ˜24 h of incubation, cells were treatedwith 0.1 mg/mL of colcemid (Invitrogen) for 4 h. Then the cells weretrypsinized, treated with hypotonic solution, and fixed. Metaphases werespread on microscope slides, and karyotype analysis was done using awell-established G banding technique. The chromosomes were classifiedaccording to the International System for Human CytogeneticNomenclature. At least 20 metaphases were analyzed per cell sample.Notably, all but one of the patients whose samples underwent karyotypicanalysis were immunosuppressed heart transplant recipients. Cytogeneticabnormalities may have been more frequent in the immunosuppressed hearttransplant patient population which predominated in the samples studied,as immunosuppressed post-transplant patients may be predisposed tolymphomas with clonal chromosomal abnormalities. Notably, in theimmunocompetent population studied in CADUCEUS, only 1 of 13clinical-production samples to date has been abnormal by cytogeneticscreening (unpublished results). However, based on the reduction ofchromosomal abnormalities in 5% O₂ culture according to severalembodiments, disclosed herein, cytogenetic screening may no longer berequired as a clinical-grade product release criterion. In someembodiments, reduction in chromosomal abnormalities due to culturing inphysiological oxygen concentrations results, regardless of the immunestatus of the subject from whom the original tissue samples werecollected. Advantageously, this broadens the scope of individuals whocan serve as tissue donors for generation of cardiac stem cells. In someembodiments, an immunocompromised transplant patient can serve as adonor for cells for use in autologous therapy (e.g., expanded cells tobe administered to that patient) or even for allogeneic therapy.

Flow Cytometry

To investigate how physiological low-oxygen culture affects thesubpopulation of c-kit⁺ stem cells and the expression of p16INK4A, amarker for cell senescence, CDCs expanded in 5% O2 and 20% O2 wereharvested as single-cell suspensions using trypsin digestion. Cells werethen incubated with PE-conjugated mouse anti-human c-kit antibody(eBioscience) or mouse anti-human p16INK4A antibody (BD Biosciences) for60 min. The expressions of c-kit and p16INK4A were quantitativelymeasured using a FACS Calibur flow cytometer with CellQuest software (BDBiosciences).

Immunostaining

To determine the myogenic differentiation in vitro, CDCs were seeded onfibronectin-coated four-chamber culture slides and continuously culturedin 5% O₂ or 20% O₂. After 3 days of culture, cells were fixed, blockedwith goat serum for 30 min, and then incubated with mouse anti-humantroponin T antibody (R&D Systems Inc.) for 1 h at room temperature.Culture slides were washed and then incubated with a PE-conjugatedsecondary antibody. Cell nuclei were stained with DAPI. Myogenicdifferentiation was quantified by calculating the positive-stained areausing the Image-Pro Plus software (version 5.1.2, Media CyberneticsInc., Carlsbad, Calif., USA).

The telomerase activity and DNA damage in CDCs were also estimated byimmunostaining with mouse anti-human telomerase catalytic subunit (TERT)antibody (Lifespan Bioscience) or rabbit polyclonal antibody againstγ-H₂AX (phosphor 5139, Abcam Inc.), as described above. Positivelystained cells were counted by fluorescence microscopy.

Senescence-Associated Beta-Galactosidase Staining

Third-passage CDCs were seeded on fibronectin-coated four-chamberculture slides and continuously cultured in 5% O₂ or 20% O₂. After 3days of culture, senescence-associated beta-galactosidase (SA-β-Gal) wasperformed according to established techniques. The SA-β-Gal-positivecells were counted under a microscope.

TUNEL Assay

To quantify the resistance to oxidative stress in vitro, CDCs wereseeded on fibronectin-coated four-chamber culture slides andcontinuously cultured in 5% O₂ or 20% O₂. After 2 days of culture, cellswere moved into a general incubator (20% O₂), and cultured with orwithout the addition of 100 mM H₂O₂ in the medium for another 24 h.Cells were fixed, and apoptotic cells were detected by TUNEL assay(Roche Diagnostics, Mannheim, Germany), according to the manufacturer'sinstructions. Cell nuclei were stained with DAPI, and TUNEL-positivecells were counted under fluorescence microscopy.

Measurement of Intracellular Reactive Oxygen Species

To measure intracellular reactive oxygen species (ROS), CDCs were seededin 6- or 96-well plates coated with 20 mg/mL of fibronectin andcontinuously cultured in 5% O₂ or 20% O₂. At ˜90% confluence, cells wereincubated with 10 mM 2′,7-dichlorodihydrofluorescein diacetate (DCF;Invitrogen) for 60 min. The fluorescence of2′,7′-dichlorodihydrofluorescein in cells cultured in 96-well plates isdirectly determined using SpectraMax M5 (Molecular Devices Corp.) withan excitation wavelength of 495 nm and an emission wavelength of 520 nm.Cells cultured in six-well plates were trypsin-treated and fixed. TheDCF fluorescence intensity in cells was analysed by flow cytometry asdescribed above.

Western Blotting

Western blot analysis was performed to compare the expressions ofintegrin-α₂, laminin-β₁, and c-Myc in CDCs cultured under 5% O₂ and 20%O₂. The equivalent of 30 mg of total protein was loaded onto SDS-PAGEgels, and then transferred to PVDF membranes. After overnight blocking,membranes were incubated with mouse anti-human integrin-α₂ antibody,mouse anti-human laminin-β₁ antibody, rabbit anti-β-actin monoclonalantibody (Lifespan Bioscience), or mouse anti-human c-Myc antibody (BDBiosciences). The appropriate horseradish peroxidase-conjugatedsecondary antibodies were used, and then the blots were visualized byusing SuperSignal West Femto maximum sensitivity substrate (ThermoScientific) and exposed to Gel Doc™ XR System (Bio-Rad Lab. Inc.).Quantitative analysis for blots was done by Quantity One software, andexpressions were normalized by b-actin.

ELISA

To compare the potency of the productions of growth factors, CDCsexpanded under 5% O₂ or 20% O₂ were seeded in 24-well culture plates ata density of 1×10⁵ mL and incubated for 3 days with hypoxic stimulation(under 1% O₂ to mimic the microenvironment of the ischemic heart). Thesupernatants were collected and the concentrations of Angiopoietin-2,bFGF, HGF, IGF-1, SDF-1, and VEGF were measured with human ELISA kits(R&D Systems Inc.), according to the manufacturer's instructions.

In Vitro Angiogenesis Assay

Angiogenic potency was estimated by tube formation using an in vitroangiogenesis assay kit (Chemicon Int.), according to the manufacturer'sinstructions. Briefly, CDCs expanded under 5% O₂ or 20% O₂ were seededon ECMatrix™-coated 96-well plates at a density of 2×10⁴ cells per well.After 6 h incubation with hypoxic stimulation (under 1% O₂ to mimic themicroenvironment of ischemic heart), tube formation were imaged. Thetotal tube length was then measured with Image-Pro Plus software.

Myocardial Infarction Model and Cell Implantation

An acute myocardial infarction was created in adult male SCID-beige mice(10-12 weeks old), as described above. The study was approved by theInstitutional Animal Care and Use Committee of Cedars-Sinai MedicalCenter and conformed to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health (NIHPublication No. 85-23, revised 1996). Briefly, after general anaesthesiaand tracheal intubation, mice were artificially ventilated with roomair. A left thoracotomy was performed through the fourth intercostalspace and the left anterior descending artery was ligated with 9-0prolene under direct vision. The mice were then randomized and subjectedto intramyocardial injections with a 30 G needle at four points in theinfarct border zone with 40 mL of PBS (PBS group, n=10), 1×10⁵ CDCsexpanded under 20% O₂ (20% O₂ CDCs, n=10), or 1×10⁵ CDCs expanded under5% O₂ (5% O₂ CDCs, n=12).

Quantification of Engraftment by Real-Time PCR

Quantitative PCR was performed 24 h and 7 days after cell injection insix to seven animals from each cell-injected group to quantify cellretention/engraftment. Male human CDCs were injected to enable detectionof the SRY gene located on the Y chromosome. The whole mouse heart washarvested, weighed, and homogenized. The TaqMan assay was used toquantify the number of transplanted cells with the human SRY gene astemplate (Applied Biosystems, CA, USA). A standard curve was generatedwith multiple dilutions of genomic DNA isolated from the injected CDCsto quantify the absolute gene copy numbers. All samples were spiked withequal amounts of genomic DNA from non-injected mouse hearts as control.For each reaction, 50 ng of genomic DNA was used. Real-time PCR wasperformed with an Applied Biosystems 7900 HT Fast real-time PCR machine.Experiments were performed in triplicate. The number of engrafted cellsper heart was quantified by calculating the copy number of SRY gene inthe total amount of DNA based on the standard curve.

Echocardiography

Mice underwent echocardiography at 3 h (baseline) and 3 weeks aftersurgery using Vevo 770™ Imaging System (VISUALSONICS™, Toronto, Canada).After the induction of light general anaesthesia, the hearts were imagedtwo-dimensionally (2D) in long-axis views at the level of the greatestLV diameter. LV end diastolic volume, LV end systolic volume, and LVejection fraction (LVEF) were measured with VisualSonics V1.3.8 softwarefrom 2D long-axis views taken through the infarcted area.

Histology

Mice were sacrificed 3 weeks after treatment. Hearts were sectioned in 5mm slices and fixed with 4% paraformaldehyde. The engraftment ofimplanted human CDCs was identified by immunostaining with antihumannuclear antigen (HNA) antibody (Chemicon). To measure cell engraftment,10 images of the infarction and border zones were selected randomly fromeach animal (three sections/animal; 1 mm separation between sections;20× magnification, Eclipse TE2000-U). The percentages of human nucleiper total nuclei were quantified using Image-Pro Plus software (version5.1.2, Media Cybernetics Inc.), and the average value from each heartwas used for statistical analysis.24 The differentiation of cardiac stemcells into myocytes, smooth muscle cells, and endothelial cells wasidentified by immunostaining with monoclonal antibodies againsthuman-specific α-sarcomeric actin, smooth muscle actin, and vonWillebrand factor (vWF) (Sigma), respectively, as described above.Masson's trichrome staining was also performed to examine the infarctionsize, LV wall thickness, LV chamber area, and viable myocardium, asdescribed previously.

Statistical Analysis

All results are presented as mean±SD. Statistical significance betweentwo groups was determined using the two-tailed paired t-test and amonggroups by ANOVA followed by Bonferroni post hoc test (SPSS II, Chicago,Ill., USA), unless otherwise indicated. Differences were consideredstatistically significant when P<0.05.

Results Cell Production

Compared with culture in 20% O₂, the outgrowth of cells from the‘explants’ was faster in 5% O₂, resulting in more than two-fold higheryield of outgrowth cells (P, 0.001, FIG. 18A). The proliferation of CDCswas similar at subsequent early passages, although proliferation atlater passages was higher in CDCs expanded in 5% O₂ (P<0.05, FIG. 18B).In several embodiments, the increased proliferation is due to inductionof HIF-1α (or other hypoxia-mediated factors) and subsequent adaptationof the cells to the reduced oxygen. In some embodiments, 5% O₂ cultureconditions increases cell migration out from the ‘explants’ through theinduction of HIF-la (or other hypoxia-mediated factors). Interestingly,the degree of hypoxia in 5% O₂ culture was insufficient to increase theconcentrations of certain chemokines and cytokines in the media,including VEGF and SDF-1, (data not shown). Thus in some embodiments,increased proliferation of cardiac stem cells occurs in amigratory-independent fashion (e.g., in the absence of HIF-1α (or otherhypoxia-mediated factors such as VEGF or SDF-1). In several embodiments,increased proliferation is due to hypoxia induced increases in cellcycle entry, or reduced apoptosis, or a combination of these mechanisms(and/or in combination with other mechanisms disclosed herein).

Other studies have suggested that culturing cells in physiologicallow-oxygen culture increases the ‘stemness’ of cells, for exampleembryonic stem cells. However, according to the presently disclosedmethods and compositions, the c-kit+ subpopulation and differentiationpotency were not augmented in these twice-passaged CDCs expanded in 5%O₂ relative to 20% O₂. Furthermore, the expression levels ofintegrin-α₂, laminin-β₁, and c-Myc were also comparable in the twoconditions. Thus, in some embodiments, cardiac stem cells respond in adifferent way to physiologic oxygen concentrations as compared to othertypes of stem cells. These differences likely reflect differences in theresponses of specific cell types to hypoxia. In some embodiments, suchdifferences are advantageous, the differential responses of differentcell types are used to optimize the reproduction (e.g., expansion),viability, engraftment, and/or functional effect of a population ofcells to be administered to a certain patient. For example, differentdegrees of hypoxia (e.g., a sliding scale of oxygen concentrations)impact cardiac stem cells differently, in some embodiments. In someembodiments, cardiac stem cells isolated from highly oxygenated regionsof the cardiac tissue respond differently to oxygen concentrations thatdiffer from their in vivo micro-environment. As a non-limiting example,a cardiac stem cell that originated from cardiac tissue exposed to a 5%concentration of oxygen in vivo was exposed to a 3% concentration inculture, the resultant expanded population may proliferate slightlyless, but provide more efficient engraftment as compared to the samecells cultured in 5% oxygen.

Chromosomal Abnormalities

Consistent with the preliminary karyotyping data discussed above, 6 of16 (37.5%) CDC samples expanded in 20% O₂ included aneuploid cells (26of 323 examined cells, FIG. 19A). The most common changes were trisomy 8(FIG. 24) and Y chromosome loss. In contrast, in 16 CDC samples expandedunder 5% O₂, there was only one aneuploid cell (from a total of 321examined cells; P<0.01 by χ² test, FIG. 19A). The aneuploidy herereflected loss of the Y chromosome, an innocuous cytogeneticabnormality. These differences in genomic stability cannot be due todifferences in the source tissue, as the same biopsies were subdividedand cultured in parallel to facilitate direct comparison. Thepercentages of aneuploid cells were also lower in CDCs expanded under 5%O₂ than in 20% O₂ (P=0.028 by unpaired t-test, FIG. 19B). Thus, inseveral embodiments, ex vivo expansion of human cardiac stem cells underphysiological low oxygen conditions (e.g., about 5% O₂) reduces theincidence of chromosomal abnormalities. In several embodiments, oxygenconcentrations are tailored specifically to the region of tissue fromwhich the original cardiac cells were collected and subsequentlyexpanded. For example, if cells were collected from the interior wall ofthe heart, which is frequently exposed to oxygenated blood, an oxygenconcentration that mimics this in vivo environment may be used toculture cardiac stem cells collected from this region. In otherembodiments, cells are collected from a surgical sample, in particularfrom within the myocardial wall, a lower oxygen concentration may beused during several embodiments, of the expansion protocol. In severalembodiments, oxygen concentrations are tailored specifically to theregion of tissue to be treated. For example, if a subject has damagedcardiac tissue on the interior wall of the heart, which is frequentlyexposed to oxygenated blood, a first oxygen concentration may be used toculture cardiac stem cells to treat this region. In other embodiments,if damaged tissue is within the myocardial wall, a lower oxygenconcentration may be used.

Phenotype and In Vitro Myogenic Differentiation

The subpopulation of c-kit+ stem cells was comparable in CDCs expandedin 5% O₂ and in 20% O₂ (P=0.106 FIGS. 25A and 25C). Likewise, thepropensity for cardiomyogenic differentiation, by immunostaining forhuman-specific cardiac troponin T, was equivalent in the two conditions(FIGS. 25B and 25D). Thus, in some embodiments, the use of physiologicaloxygen concentrations does not alter the “stemness” profile of thecultured cells, at least with regard to expression of certain markers(e.g., c-kit). In other embodiments, however, alterations in stem cellmarker profiles result from alterations in oxygen concentration. Assuch, in some embodiments, the profile of the resultant cells may becontrolled at least in part by the oxygen concentration used in theculturing process.

Cell Senescence

Cell senescence was evaluated by the expression of p16^(INK4A),telomerase activity, and SA-β-Gal staining. The expression ofp16^(INK4A) was lower (FIGS. 20A and 20D, P<0.001), and the telomeraseactivity (evaluated by the expression of TERT) was higher (FIGS. 20B and20E, P<0.01) in CDCs expanded in 5% O₂. The fraction ofSA-β-Gal-positive cells was also lower in third-passage CDCs expanded in5% O₂ than in 20% O₂ (FIGS. 20C and 20F, P<0.05). Thus, in someembodiments, the culture conditions and methods disclosed herein reducethe “aging” rate of cultured cells. In some embodiments, this isrealized as a decrease in expression in one or more genes (or proteins)related to the aging of a cell, cell cycle entry (or activity), tumorsuppressor genes, and the like. Moreover, in some embodiments,karyotypic abnormalities and/or aging of cells is reduced by virtue ofincrease telomerase activity (e.g., reduced telomere shortening).

Intracellular ROS, DNA Damage, and Resistance to Oxidative Stress

Intracellular ROS concentrations were measured by DCF staining.Intracellular ROS level was lower in 5% O₂-cultured CDCs than in 20%O₂-cultured CDCs (FIGS. 21A and 21B; P<0.01). The percentage of cellswith γ-H₂AX foci, a marker of DNA damage was likewise lower in 5%O₂-cultured CDCs (FIGS. 21C and 21D; P<0.01). This correlates with thekaryotyping data discussed above, which showed a dramatic reduction ofaneuploidy in CDCs expanded under 5% O₂ (see e.g., FIG. 19). Thus, inseveral embodiments, the culturing methods and compositions disclosedherein reduce the level of intracellular ROS, which in turn, reduces theincidence of karyotypic abnormalities. In some embodiments, the decreasein ROS also increases cellular viability. In some embodiments, increasedviability is manifest by increased proliferation in culture, increasedviability after in vivo administration, and/or improved engraftmentafter in vivo administration.

In order to determine whether cells cultured in physiological O₂ weremore resistant (or more sensitive), to oxidative stress, CDCs weretherefore exposed to H₂O₂, a powerful oxidant. After 24 h of exposure to100 mM H₂O₂, the number of apoptotic cells was lower in 5% O₂ CDCs thanin those grown in 20% O₂ (FIGS. 21E and 21F, P<0.01). The basalfrequency of apoptosis was also lower in the 5% O₂ CDCs (P<0.05). Thus,in some embodiments, not only do the disclosed culture methods andcompositions reduce formation of ROS that naturally appear, but theyreduce the sensitivity of cells to sources of oxidative stress. In someembodiments, as a result, cells are more robust in a wider variety ofenvironments in vivo, and thus, have greater viability.

Expression of Adhesion Molecules and c-Myc

The expression levels of integrin-α₂, laminin-β₁, and c-Myc werecomparable in CDCs expanded in 5% O₂ and in 20% O₂ (see FIGS. 26A-26D).

In Vitro Production of Growth Factors and Tube Formation

While CDCs are known to secrete a variety of growth factors the presentstudy analysed conditioned media to determine whether hypoxic cultureinfluences the paracrine secretion of selected growth factors by CDCs.Cells cultured either in 5% O₂ or 20% O₂ were plated in fresh media andgrown for 3 days with hypoxic stimulation (1% O₂) to mimic themicroenvironment of ischemic heart. The production of the majority ofgrowth factors under hypoxic stimulation, including angiopoietin-2,bFGF, HGF, IGF-1, SDF-1, and VEGF, was comparable in CDCs expanded under5% O₂ or 20% O₂ (see FIGS. 27A-27F), although some factors tended to behigher in CDCs expanded under 5% O₂.

By in vitro angiogenesis assay, CDCs expanded under both 5% O₂ and 20%O₂ could form capillary-like networks (tube formation) on matrigel in 6h with hypoxic stimulation, and there was no significant differencebetween groups (see FIGS. 28A-28B).

In several embodiments, in vivo multilineage differentiation from humanCDCs expanded under either 5% O₂ or 20% O₂, is achieved as well as therobust production of various angiogenic and anti-apoptotic growthfactors in vitro. However, several embodiments are particularlyadvantageous in that the above results are achieved without theincreased incidence of genomic instability. As such, cells culturedunder physiologic oxygen concentrations are particularly advantageousfor use in cellular therapies (e.g., cardiac stem cell therapy).

In Vivo Cell Engraftment and Differentiation

The ultimate test of cell quality for cells to be used in cardiacregeneration and repair is the efficacy of the cells in vivo. Cellretention (24 h) and engraftment (7 days) in hearts injected with CDCscultured in 5% O₂ or 20% O₂ was quantified. Quantitative PCR analysisshowed that CDCs expanded in 5% O₂ survived better than CDCs expanded in20% O₂, both at 24 h and at 7 days after implantation into the infarctedhearts of SCID mice (FIG. 22A). Furthermore, in the scar and marginalregions of the infarcted mouse heart 3 weeks after treatment, greatersurvival of CDCs expanded in 5% O₂ than in 20% O₂ was detected (FIG.22A). Quantitative analysis of percentages of human nuclei revealedbetter cell engraftment after implantation of CDCs expanded under 5% O₂than under 20% O₂ (3.40+0.94 vs. 1.77+0.52%, P<0.01, FIGS. 22B and 22C).The improvements in retention and engraftment suggest greatertransplanted cell resilience, consistent with the observed increase inresistance to oxidative stress (FIGS. 21A and 21D) and the decrease insenescence (FIG. 20) in 5% O₂-cultured CDCs. One distinctive feature ofCDCs is the ability to detect consistent cardiomyogenic and angiogenicdifferentiation of injected cells (e.g., direct regenerative effects),even though the mechanism of functional benefit appears, in someembodiments, to be largely paracrine-mediated. The present study alsoconfirmed that CDCs can differentiate into cardiomyocytes, endothelialand vascular smooth muscle cells, irrespective of the level of oxygen inwhich they were cultured. Histology of mouse hearts that had beeninjected with CDCs 3 weeks earlier revealed expression of α-sarcomericactin (see FIGS. 29A-29D), smooth muscle actin (see FIGS. 30A-30B), andvWF (see FIGS. 31A-31B) in some of the surviving progeny of human CDCsexpanded in either 5% O₂ or 20% O₂, indicative of multilineagedifferentiation. Although CDCs expanded in 5% O₂ exhibited greatersurvival, their differentiation potential was not noticeably differentthan that of 20% O₂-cultured CDCs.

When implanted into infarcted hearts of SCID mice, 5% O₂-cultured humanCDCs engrafted to a greater degree and improved function to a greaterdegree as compared to 20% O₂-cultured CDCs. As discussed above, thedecrease in cell senescence and increased resistance to oxidative stressin vitro are, in some embodiments, at least partially responsible forthe superior effects observed in vivo. Also as discussed above, it isbelieved that implantation of CDCs into infarcted heart leads tofunctional improvement via a direct regeneration, paracrine effects, ora combination thereof. Thus, in several embodiments, expansion ofcardiac stem cells in physiologic oxygen concentrations augments theability of the cells to effect cardiac repair and/or regeneration. Insome embodiments, the cells cultured in physiologic oxygenconcentrations engraft more readily than cells cultured in 20% O₂. Insome embodiments, engraftment is more efficient (e.g., a greaterproportion of administered cells engraft). In some embodiments,engraftment is for a longer term (e.g., engrafted cells cultured inphysiologic oxygen concentrations survive longer than those cultured in20% O₂. In several embodiments, increased engraftment or longer termengraftment is related to the decreased senescence of cells cultured inphysiologic oxygen concentrations (e.g., the cells are moremetabolically active and more readily survive in a new hostenvironment).

While the present study did not attempt to quantify the relative rolesof direct regeneration vs. indirect paracrine effects, in someembodiments, both direct and indirect effects are operative. Asdiscussed above, direct effects (e.g., the differentiation of theadministered cells into functional cardiac, vascular, or endothelialtissue are responsible for repair and/or regeneration of cardiac tissue,in some embodiments. In some embodiments, paracrine effects (e.g.,growth factors, cytokines, or other signals/factors) generated by theadministered cells serve to mediate cardiac tissue repair orregeneration. In some embodiments, combinations of direct and indirectmechanisms are involved.

Cardiac Function and Infarct Size

To assess efficacy in myocardial repair, left ventricular function inhearts injected with vehicle or with CDCs cultured in 5% O₂ or 20% O₂was compared. Although LVEF at baseline did not differ between groups,LEVF measured 3 weeks after treatment was higher in mice implanted with5% O₂-cultured CDCs as compared to those expanded in 20% O₂ (41.5+3.2vs. 36.5+4.1%, P=0.043, FIG. 22D). Both cell groups outperformed thevehicle-treated group. In addition, the absolute values of LVEFcorrelate strongly with the degree of cell engraftment (r²=0.80,P=0.002, FIG. 22E). Compared with the control treatment with PBSinjection only, the implantation of CDCs expanded under either 5% O₂ or20% O₂ results in obviously smaller infarct size, more viablemyocardium, increased infarct wall thickness, and lower LV chamber area3 weeks after treatment (P<0.05 vs. PBS, FIG. 23A-23G). These parametersshowed greater improvements with administration of 5% O₂-cultured CDCsas compared to those expanded in 20% O₂ (P, 0.05). Thus, as discussedabove, in several embodiments, culture of cells in physiologic oxygenconcentrations augments the ability of the cells to effect cardiacrepair. In some embodiments, use of physiologic oxygen concentrationsduring culture of cells to be administered improves the functional oranatomical endpoints (as compared to cells cultured in 20% oxygen) by atleast about 5%. In some embodiments, cells cultured in physiologicoxygen concentrations improve functional or anatomic endpoints by about5% to about 10%, about 10% to about 15%, about 15% to about 20%, andoverlapping ranges thereof. In some embodiments, depending on theendpoint, use of physiologic oxygen concentrations during culture ofcells to be administered improves the effects the therapy (e.g., therepair or regeneration of cardiac tissue) by about 2-fold, about 4-fold,about 6-fold, about 10-fold, about 20-fold, and overlapping ranges lyingbetween these values.

In several embodiments, ex vivo expansion of human CDCs underphysiological oxygen (5% O₂) results in one or more of the following:increases in cell yield, decreases in aneuploidy and cell senescence,and increases in resistance to oxidative stress. Furthermore, in someembodiments, CDCs expanded in 5% O₂ results in improved engraftment andfunctional benefit after implantation into infarcted hearts (or otherdamaged or diseased heart tissue).

Various modifications and applications of embodiments of the inventionmay be performed without departing from the true spirit or scope of theinvention. Method steps disclosed herein need not be performed in theorder set forth. It should be understood that the invention is notlimited to the embodiments set forth herein for purposes ofexemplification, but is to be defined only by a reading of the appendedclaims, including the full range of equivalency to which each elementthereof is entitled.

What is claimed is:
 1. A method for reducing the incidence of karyotypicabnormalities in cardiac stem cells for use in the repair orregeneration of cardiac tissue, the method comprising: isolating cardiacstem cells, wherein said cells are isolated from healthy mammaliannon-embryonic cardiac tissue; and culturing said isolated stem cells ina culture media supplemented with an antioxidant composition, whereinsaid antioxidant composition comprises at least one peptide antioxidantand at least one non-peptide antioxidant, wherein said at least onepeptide antioxidant is present in a concentration ranging from about 0.1to 200 μM, wherein said at least at least one non-peptide antioxidant ispresent in a concentration ranging from about 0.1 to 200 μM, whereinsaid antioxidant composition reduces reactive oxygen species to a levelwhich decreases oxidative-stress induced DNA damage, wherein saidreduction in reactive oxygen species does not significantly reducemarkers of DNA repair mechanisms, wherein said reduction in reactiveoxygen species does not significantly induce markers of DNA damage insaid cardiac stem cells, and wherein the balance of reduced reactiveoxygen species, non-reduced DNA repair mechanisms and non-induction ofDNA damage reduces the incidence of karyotypic abnormalities in saidcardiac stem cells.